Robert J. Schmitz

Widespread dynamic DNA methylation in response to biotic stress

Regulation of gene expression by DNA methylation is crucial for defining cellular identities and coordinating organism-wide developmental programs in many organisms. In plants, modulation of DNA methylation in response to environmental conditions represents a potentially robust mechanism to regulate gene expression networks; however, examples of dynamic DNA methylation are largely limited to gene imprinting. Here we report an unexpected role for DNA methylation in regulation of the Arabidopsis thaliana immune system. Profiling the DNA methylomes of plants exposed to bacterial pathogen, avirulent bacteria, or salicylic acid (SA) hormone revealed numerous stress-induced differentially methylated regions, many of which were intimately associated with differentially expressed genes. In response to SA, transposon-associated differentially methylated regions, which were accompanied by up-regulation of 21-nt siRNAs, were often coupled to transcriptional changes of the transposon and/or the proximal gene. Thus, dynamic DNA methylation changes within repetitive sequences or transposons can regulate neighboring genes in response to SA stress.

Transgenerational Epigenetic Instability Is a Source of Novel Methylation Variants

Spontaneous methylation rates that may affect phenotype in the plant Arabidopsis are higher than the mutation rate. Epigenetic information, which may affect an organism’s phenotype, can be stored and stably inherited in the form of cytosine DNA methylation. Changes in DNA methylation can produce meiotically stable epialleles that affect transcription and morphology, but the rates of spontaneous gain or loss of DNA methylation are unknown. We examined spontaneously occurring variation in DNA methylation in Arabidopsis thaliana plants propagated by single-seed descent for 30 generations. We identified 114,287 CG single methylation polymorphisms and 2485 CG differentially methylated regions (DMRs), both of which show patterns of divergence compared with the ancestral state. Thus, transgenerational epigenetic variation in DNA methylation may generate new allelic states that alter transcription, providing a mechanism for phenotypic diversity in the absence of genetic mutation.

Patterns of population epigenomic diversity.

Natural epigenetic variation provides a source for the generation of phenotypic diversity, but to understand its contribution to such diversity, its interaction with genetic variation requires further investigation. Here we report population-wide DNA sequencing of genomes, transcriptomes and methylomes of wild Arabidopsis thaliana accessions. Single cytosine methylation polymorphisms are not linked to genotype. However, the rate of linkage disequilibrium decay amongst differentially methylated regions targeted by RNA-directed DNA methylation is similar to the rate for single nucleotide polymorphisms. Association analyses of these RNA-directed DNA methylation regions with genetic variants identified thousands of methylation quantitative trait loci, which revealed the population estimate of genetically dependent methylation variation. Analysis of invariably methylated transposons and genes across this population indicates that loci targeted by RNA-directed DNA methylation are epigenetically activated in pollen and seeds, which facilitates proper development of these structures.

Processing and Subcellular Trafficking of ER-Tethered EIN2 Control Response to Ethylene Gas

Cleave and Leave Plants produce ethylene gas, which acts as a hormone and is essential for the ripening of fruit, the resistance of plants to pathogens, the adaptation of plants to stress conditions, and stem cell maintenance. Although many components of the ethylene gas signaling pathway have been well studied, little is known about how the ethylene receptors located in the endoplasmic reticulum (ER) membrane can transmit the signal to the nucleus. Studying Arabidopsis, Qiao et al. (p. 390, published online 30 August) found that perception of ethylene gas in the ER promotes signal transduction via cleavage and rapid ER-nucleus translocation of the cytosolic portion of the transmembrane ETHYLENE INSENSITIVE2 protein, which activates ethylene-dependent gene expression and other ethylene response phenotypes in plants. The plant hormone ethylene triggers cleavage and translocation to the nucleus of a signaling component. Ethylene gas is essential for many developmental processes and stress responses in plants. ETHYLENE INSENSITIVE2 (EIN2), an NRAMP-like integral membrane protein, plays an essential role in ethylene signaling, but its function remains enigmatic. Here we report that phosphorylation-regulated proteolytic processing of EIN2 triggers its endoplasmic reticulum (ER)–to–nucleus translocation. ER-tethered EIN2 shows CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) kinase–dependent phosphorylation. Ethylene triggers dephosphorylation at several sites and proteolytic cleavage at one of these sites, resulting in nuclear translocation of a carboxyl-terminal EIN2 fragment (EIN2-C′). Mutations that mimic EIN2 dephosphorylation, or inactivate CTR1, show constitutive cleavage and nuclear localization of EIN2-C′ and EIN3 and EIN3-LIKE1–dependent activation of ethylene responses. These findings uncover a mechanism of subcellular communication whereby ethylene stimulates phosphorylation-dependent cleavage and nuclear movement of the EIN2-C′ peptide, linking hormone perception and signaling components in the ER with nuclear-localized transcriptional regulators.

Linking photoreceptor excitation to changes in plant architecture

Plants sense neighbor proximity as a decrease in the ratio of red to far-red light, which triggers a series of developmental responses. In Arabidopsis, phytochrome B (PHYB) is the major sensor of shade, but PHYB excitation has not been linked directly to a growth response. We show that the basic helix-loop-helix (bHLH) transcription factor PIF7 (phytochrome-interacting factor 7), an interactor of PHYB, accumulates in its dephosphorylated form in shade, allowing it to bind auxin biosynthetic genes and increase their expression. New auxin synthesized through a PIF7-regulated pathway is required for shade-induced growth, linking directly the perception of a light quality signal to a rapid growth response.

RF1 knockout allows ribosomal incorporation of unnatural amino acids at multiple sites

Stop codons have been exploited for genetic incorporation of unnatural amino acids (Uaas) in live cells, but the efficiency is low possibly due to competition from release factors, limiting the power and scope of this technology. Here we show that the reportedly essential release factor 1 can be knocked out from Escherichia coli by fixing release factor 2. The resultant strain JX33 is stable and independent, and reassigns UAG from a stop signal to an amino acid when a UAG-decoding tRNA/synthetase pair is introduced. Uaas were efficiently incorporated at multiple UAG sites in the same gene without translational termination in JX33. We also found that amino acid incorporation at endogenous UAG codons is dependent on RF1 and mRNA context, which explains why E. coli tolerates apparent global suppression of UAG. JX33 affords a unique autonomous host for synthesizing and evolving novel protein functions by enabling Uaa incorporation at multiple sites.

1,135 Genomes Reveal the Global Pattern of Polymorphism in Arabidopsis thaliana

Summary Arabidopsis thaliana serves as a model organism for the study of fundamental physiological, cellular, and molecular processes. It has also greatly advanced our understanding of intraspecific genome variation. We present a detailed map of variation in 1,135 high-quality re-sequenced natural inbred lines representing the native Eurasian and North African range and recently colonized North America. We identify relict populations that continue to inhabit ancestral habitats, primarily in the Iberian Peninsula. They have mixed with a lineage that has spread to northern latitudes from an unknown glacial refugium and is now found in a much broader spectrum of habitats. Insights into the history of the species and the fine-scale distribution of genetic diversity provide the basis for full exploitation of A. thaliana natural variation through integration of genomes and epigenomes with molecular and non-molecular phenotypes.

The genome sequences of Arachis duranensis and Arachis ipaensis , the diploid ancestors of cultivated peanut

Cultivated peanut (Arachis hypogaea) is an allotetraploid with closely related subgenomes of a total size of ∼2.7 Gb. This makes the assembly of chromosomal pseudomolecules very challenging. As a foundation to understanding the genome of cultivated peanut, we report the genome sequences of its diploid ancestors (Arachis duranensis and Arachis ipaensis). We show that these genomes are similar to cultivated peanuts A and B subgenomes and use them to identify candidate disease resistance genes, to guide tetraploid transcript assemblies and to detect genetic exchange between cultivated peanuts subgenomes. On the basis of remarkably high DNA identity of the A. ipaensis genome and the B subgenome of cultivated peanut and biogeographic evidence, we conclude that A. ipaensis may be a direct descendant of the same population that contributed the B subgenome to cultivated peanut.

Temporal transcriptional response to ethylene gas drives growth hormone cross-regulation in Arabidopsis

The gaseous plant hormone ethylene regulates a multitude of growth and developmental processes. How the numerous growth control pathways are coordinated by the ethylene transcriptional response remains elusive. We characterized the dynamic ethylene transcriptional response by identifying targets of the master regulator of the ethylene signaling pathway, ETHYLENE INSENSITIVE3 (EIN3), using chromatin immunoprecipitation sequencing and transcript sequencing during a timecourse of ethylene treatment. Ethylene-induced transcription occurs in temporal waves regulated by EIN3, suggesting distinct layers of transcriptional control. EIN3 binding was found to modulate a multitude of downstream transcriptional cascades, including a major feedback regulatory circuitry of the ethylene signaling pathway, as well as integrating numerous connections between most of the hormone mediated growth response pathways. These findings provide direct evidence linking each of the major plant growth and development networks in novel ways. DOI: http://dx.doi.org/10.7554/eLife.00675.001

Targeted genome modifications in soybean with CRISPR/Cas9

BackgroundThe ability to selectively alter genomic DNA sequences in vivo is a powerful tool for basic and applied research. The CRISPR/Cas9 system precisely mutates DNA sequences in a number of organisms. Here, the CRISPR/Cas9 system is shown to be effective in soybean by knocking-out a green fluorescent protein (GFP) transgene and modifying nine endogenous loci.ResultsTargeted DNA mutations were detected in 95% of 88 hairy-root transgenic events analyzed. Bi-allelic mutations were detected in events transformed with eight of the nine targeting vectors. Small deletions were the most common type of mutation produced, although SNPs and short insertions were also observed. Homoeologous genes were successfully targeted singly and together, demonstrating that CRISPR/Cas9 can both selectively, and generally, target members of gene families. Somatic embryo cultures were also modified to enable the production of plants with heritable mutations, with the frequency of DNA modifications increasing with culture time. A novel cloning strategy and vector system based on In-Fusion® cloning was developed to simplify the production of CRISPR/Cas9 targeting vectors, which should be applicable for targeting any gene in any organism.ConclusionsThe CRISPR/Cas9 is a simple, efficient, and highly specific genome editing tool in soybean. Although some vectors are more efficient than others, it is possible to edit duplicated genes relatively easily. The vectors and methods developed here will be useful for the application of CRISPR/Cas9 to soybean and other plant species.