Summary: Ethylene is an important plant growth regulator perceived by membrane‐bound ethylene receptors. The ETR1 ethylene receptor is positively regulated by a predicted membrane protein, RTE1, based on genetic studies in Arabidopsis. RTE1 homologs exist in plants, animals and protists, but the molecular function of RTE1 is unknown. Here, we examine RTE1 expression and subcellular protein localization in order to gain a better understanding of RTE1 and its function in relation to ETR1. Arabidopsis plants transformed with the RTE1 promoter fused to the β‐glucuronidase (GUS) reporter gene revealed that RTE1 expression partly correlates with previously described sites of ETR1 expression or sites of ethylene response, such as the seedling root, root hairs and apical hook. RTE1 transcript levels are also enhanced by ethylene treatment, and reduced by the inhibition of ethylene signaling. For subcellular localization of RTE1, a functional RTE1 fusion to red fluorescent protein (RFP) was expressed under the control of the native RTE1 promoter. Using fluorescence microscopy, RTE1 was observed primarily at the Golgi apparatus and partially at the endoplasmic reticulum (ER) in stably transformed Arabidopsis protoplasts, roots and root hairs. Next, a functional ETR1 fusion to a 5xMyc epitope tag was expressed under the control of the native ETR1 promoter. Immunohistochemistry of root hairs not only showed ETR1 residing at the ER as previously reported, but revealed substantial localization of ETR1 at the Golgi apparatus. Lastly, we demonstrated the subcellular co‐localization of RTE1 and ETR1. These findings support and enhance the genetic model that RTE1 plays a role in regulating ETR1.

Background: Nucleosomes are the building blocks of chromatin where gene regulation takes place. Chromatin landscapes have been profiled for several species, providing insights into the fundamental mechanisms of chromatin-mediated transcriptional regulation of gene expression. However, knowledge is missing for several major and deep-branching eukaryotic groups, such as the Stramenopiles, which include the diatoms. Diatoms are highly diverse and ubiquitous species of phytoplankton that play a key role in global biogeochemical cycles. Dissecting chromatin-mediated regulation of genes in diatoms will help understand the ecological success of these organisms in contemporary oceans. Results: Here, we use high resolution mass spectrometry to identify a full repertoire of post-translational modifications on histones of the marine diatom Phaeodactylum tricornutum, including eight novel modifications. We map five histone marks coupled with expression data and show that P. tricornutum displays both unique and broadly conserved chromatin features, reflecting the chimeric nature of its genome. Combinatorial analysis of histone marks and DNA methylation demonstrates the presence of an epigenetic code defining activating or repressive chromatin states. We further profile three specific histone marks under conditions of nitrate depletion and show that the histone code is dynamic and targets specific sets of genes. Conclusions: This study is the first genome-wide characterization of the histone code from a stramenopile and a marine phytoplankton. The work represents an important initial step for understanding the evolutionary history of chromatin and how epigenetic modifications affect gene expression in response to environmental cues in marine environments.

DNA cytosine methylation is a widely conserved epigenetic mark in eukaryotes that appears to have critical roles in the regulation of genome structure and transcription. Genome-wide methylation maps have so far only been established from the supergroups Archaeplastida and Unikont. Here we report the first whole-genome methylome from a stramenopile, the marine model diatom Phaeodactylum tricornutum. Around 6% of the genome is intermittently methylated in a mosaic pattern. We find extensive methylation in transposable elements. We also detect methylation in over 320 genes. Extensive gene methylation correlates strongly with transcriptional silencing and differential expression under specific conditions. By contrast, we find that genes with partial methylation tend to be constitutively expressed. These patterns contrast with those found previously in other eukaryotes. By going beyond plants, animals and fungi, this stramenopile methylome adds significantly to our understanding of the evolution of DNA methylation in eukaryotes.

Castor bean is an important oil-producing plant in the Euphorbiaceae family. Its high-quality oil contains up to 90% of the unusual fatty acid ricinoleate, which has many industrial and medical applications. Castor bean seeds also contain ricin, a highly toxic Type 2 ribosome-inactivating protein, which has gained relevance in recent years due to biosafety concerns. In order to gain knowledge on global genetic diversity in castor bean and to ultimately help the development of breeding and forensic tools, we carried out an extensive chloroplast sequence diversity analysis. Taking advantage of the recently published genome sequence of castor bean, we assembled the chloroplast and mitochondrion genomes extracting selected reads from the available whole genome shotgun reads. Using the chloroplast reference genome we used the methylation filtration technique to readily obtain draft genome sequences of 7 geographically and genetically diverse castor bean accessions. These sequence data were used to identify single nucleotide polymorphism markers and phylogenetic analysis resulted in the identification of two major clades that were not apparent in previous population genetic studies using genetic markers derived from nuclear DNA. Two distinct sub-clades could be defined within each major clade and large-scale genotyping of castor bean populations worldwide confirmed previously observed low levels of genetic diversity and showed a broad geographic distribution of each sub-clade.

The plant hormone ethylene plays important roles in growth and development. Ethylene is perceived by a family of membrane-bound receptors that actively repress ethylene responses. When the receptors bind ethylene, their signaling is shut off, activating responses. REVERSION-TO-ETHYLENE SENSITIVITY (RTE1) encodes a novel membrane protein conserved in plants and metazoans. Genetic analyses in Arabidopsis thaliana suggest that RTE1 promotes the signaling state of the ethylene receptor ETR1 through the ETR1 N-terminal domain. RTE1 and ETR1 have been shown to co-localize to the endoplasmic reticulum (ER) and Golgi apparatus in Arabidopsis. Here, we demonstrate a physical association of RTE1 and ETR1 using in vivo and in vitro methods. Interaction of RTE1 and ETR1 was revealed in vivo by bimolecular fluorescence complementation (BiFC) in a tobacco cell transient assay and in stably transformed Arabidopsis. The association was also observed using a truncated version of ETR1 comprising the N terminus (amino acids 1–349). Interaction of RTE1 and ETR1 was confirmed by co-immunoprecipitation from Arabidopsis. The interaction occurs with high affinity (K_d, 117 nm) based on tryptophan fluorescence spectroscopy using purified recombinant RTE1 and a tryptophan-less version of purified recombinant ETR1. An amino acid substitution (C161Y) in RTE1 that is known to confer an ETR1 loss-of-function phenotype correspondingly gives a nearly 12-fold increase in the dissociation constant (K_d, 1.38 μm). These findings indicate that a high affinity association of RTE1 and ETR1 is important in the regulation of ETR1.

Summary: Ethylene is an important regulator of plant growth, development and responses to environmental stresses. Arabidopsis perceives ethylene through five homologous receptors that negatively regulate ethylene responses. RTE1, a novel gene conserved in plants, animals and some protists, was recently identified as a positive regulator of the ETR1 ethylene receptor. Here, we genetically analyze the dependence of ETR1 on RTE1 in order to obtain further insight into RTE1 function. The function of RTE1 was found to be independent and distinct from that of RAN1, which encodes a copper transporter required for ethylene receptor function. We tested the ability of an rte1 loss‐of‐function mutation to suppress 11 etr1 ethylene‐binding domain mis‐sense mutations, all of which result in dominant ethylene insensitivity due to constitutive signaling. This suppression test uncovered two classes of etr1 mutations –RTE1‐dependent and RTE1‐independent. The nature of these mutations suggests that the ethylene‐binding domain is a possible target of RTE1 action. Based on these findings, we propose that RTE1 promotes ETR1 signaling through a conformational effect on the ethylene‐binding domain.