Loss or gain of DNA methylation can affect gene expression and is sometimes transmitted across generations. Such epigenetic alterations are thus a possible source of heritable phenotypic variation in the absence of DNA sequence change. However, attempts to assess the prevalence of stable epigenetic variation in natural and experimental populations and to quantify its impact on complex traits have been hampered by the confounding effects of DNA sequence polymorphisms. To overcome this problem as much as possible, two parents with little DNA sequence differences, but contrasting DNA methylation profiles, were used to derive a panel of epigenetic Recombinant Inbred Lines (epiRILs) in the reference plant Arabidopsis thaliana. The epiRILs showed variation and high heritability for flowering time and plant height (∼30%), as well as stable inheritance of multiple parental DNA methylation variants (epialleles) over at least eight generations. These findings provide a first rationale to identify epiallelic variants that contribute to heritable variation in complex traits using linkage or association studies. More generally, the demonstration that numerous epialleles across the genome can be stable over many generations in the absence of selection or extensive DNA sequence variation highlights the need to integrate epigenetic information into population genetics studies.
This document provides supplementary guidance on specific topics for the allergenicity risk assessment of genetically modified plants. In particular, it supplements general recommendations outlined in previous EFSA GMO Panel guidelines and Implementing Regulation (EU) No 503/2013. The topics addressed are non-IgE-mediated adverse immune reactions to foods, in vitro protein digestibility tests and endogenous allergenicity. New scientific and regulatory developments regarding these three topics are described in this document. Considerations on the practical implementation of those developments in the risk assessment of genetically modified plants are discussed and recommended, where appropriate. (C) 2017 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority
In yeast and animals, the anaphase-promoting complex or cyclosome (APC/C) is an essential ubiquitin protein ligase that regulates mitotic progression and exit by controlling the stability of cell cycle regulatory proteins, such as securin and the mitotic cyclins. In plants, the function, regulation, and substrates of the APC/C are poorly understood. To gain more insight into the roles of the plant APC/C, we characterized at the molecular level one of its subunits, APC2, which is encoded by a single-copy gene in Arabidopsis. We show that the Arabidopsis gene is able to partially complement a budding yeast apc2 ts mutant. By yeast two-hybrid assays, we demonstrate an interaction of APC2 with two other APC/C subunits: APC11 and APC8/CDC23. A reverse-genetic approach identified Arabidopsis plants carrying T-DNA insertions in the APC2 gene. apc2 null mutants are impaired in female megagametogenesis and accumulate a cyclin- -glucuronidase reporter protein but do not display metaphase arrest, as observed in other systems. The APC2 gene is expressed in various plant organs and does not seem to be cell cycle regulated. Finally, we report intriguing differences in APC2 protein subcellular localization compared with that in other systems. Our observations support a conserved function of the APC/C in plants but a different mode of regulation.
SUMMARYIncreased phenotyping accuracy and throughput are necessary to improve our understanding of quantitative variation and to be able to deconstruct complex traits such as those involved in growth responses to the environment. Still, only a few facilities are known to handle individual plants of small stature for non-destructive, real-time phenotype acquisition from plants grown in precisely adjusted and variable experimental conditions. Here, we describe Phenoscope, a high-throughput phenotyping platform that has the unique feature of continuously rotating 735 individual pots over a table. It automatically adjusts watering and is equipped with a zenithal imaging system to monitor rosette size and expansion rate during the vegetative stage, with automatic image analysis allowing manual correction. When applied to Arabidopsis thaliana, we show that rotating the pots strongly reduced micro-environmental disparity: heterogeneity in evaporation was cut by a factor of 2.5 and the number of replicates needed to detect a specific mild genotypic effect was reduced by a factor of 3. In addition, by controlling a large proportion of the micro-environmental variance, other tangible sources of variance become noticeable. Overall, Phenoscope makes it possible to perform large-scale experiments that would not be possible or reproducible by hand. When applied to a typical quantitative trait loci (QTL) mapping experiment, we show that mapping power is more limited by genetic complexity than phenotyping accuracy. This will help to draw a more general picture as to how genetic diversity shapes phenotypic variation.
The process of microtubule nucleation in plant cells is still a major question in plant cell biology. g-Tubulin is known as one of the key molecular players for microtubule nucleation in animal and fungal cells. Here, we provide genetic evidence that in Arabidopsis thaliana, g-tubulin is required for the formation of spindle, phragmoplast, and cortical microtubule arrays. We used a reverse genetics approach to investigate the role of the two Arabidopsis g-tubulin genes in plant development and in the formation of microtubule arrays. Isolation of mutants in each gene and analysis of two combinations of g-tubulin double mutants showed that the two genes have redundant functions. The first combination is lethal at the gametophytic stage. Disruption of both g-tubulin genes causes aberrant spindle and phragmoplast structures and alters nuclear division in gametophytes. The second combination of g-tubulin alleles affects late seedling development, ultimately leading to lethality 3 weeks after germination. This partially viable mutant combination enabled us to follow dynamically the effects of g-tubulin depletion on microtubule arrays in dividing cells using a green fluorescent protein marker. These results establish the central role of g-tubulin in the formation and organization of microtubule arrays in Arabidopsis.
We extensively phenotyped 6000 Arabidopsis plants with experimentally perturbed DNA methylomes as well as a diverse panel of natural accessions in a common garden. We found that alterations in DNA methylation not only caused heritable phenotypic diversity but also produced heritability patterns closely resembling those of the natural accessions. Our findings indicate that epigenetically induced and naturally occurring variation in complex traits share part of their polygenic architecture and may offer complementary adaptation routes in ecological settings.
We studied the expression of the four genes encoding 2S albumin seed storage proteins (at2S1 to at2S4) in Arabidopsis thaliana. All four genes followed similar temporal profiles throughout development, but at2S2 and at2S3 were expressed at significantly higher levels than at2S1 or at2S4. In situ hybridization showed that at2S2 to at2S4 mRNAs were present throughout the embryo, whereas at2S1 was expressed at levels similar to at2S2 and at2S3 in the embryo axis but at only insignificant levels in the cotyledons. The different members of the gene family are, thus, likely to be regulated by different combinations of cis-acting elements, but it cannot be ruled out that post-transcriptional factors play a role. We studied the effect of enlarging the gene family by introducing an extra, nearly identical gene driven by the promoter of at2S1. The data were consistent with a model in which the expression of at2S2 to at2S4 is not affected by that of at2S1, and in which, at least at low copy numbers of the introduced gene, there is no limit on the overall amount of RNA that the at2S gene family can produce.
We investigated the role of the ubiquitin proteasome system (UPS), which allows proteins to be selectively degraded, during gametophyte development in Arabidopsis thaliana. Three mutant alleles altering the UPS were isolated in the Wassilewskija (Ws) accession: they affect the Regulatory Particle 5a (RPT5a) gene, which (along with RPT5b) encodes one of the six AAAATPases of the proteasome regulatory particle. In the heterozygous state, all three mutant alleles displayed 50% pollen lethality, suggesting that RPT5a is essential for male gametophyte development. However, a fourth mutant in the Columbia (Col) accession did not display such a phenotype because the RPT5b Col allele complements the rpt5a defect in the male gametophyte, whereas the RPT5b Ws allele does not. Double rpt5a rpt5b mutants showed a complete male and female gametophyte lethal phenotype in a Col background, indicating that RPT5 subunits are essential for both gametophytic phases. Mitotic divisions were affected in double mutant gametophytes correlating with an absence of the proteasome-dependent cyclinA3 degradation. Finally, we show that RPT5b expression is highly increased when proteasome functioning is defective, allowing complementation of the rpt5a mutation. In conclusion, RPT5 subunits are not only essential for both male and female gametophyte development but also display accession-dependent redundancy and are crucial in cell cycle progression.
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