A. Harvey Millar

Human DNA methylomes at base resolution show widespread epigenomic differences

DNA cytosine methylation is a central epigenetic modification that has essential roles in cellular processes including genome regulation, development and disease. Here we present the first genome-wide, single-base-resolution maps of methylated cytosines in a mammalian genome, from both human embryonic stem cells and fetal fibroblasts, along with comparative analysis of messenger RNA and small RNA components of the transcriptome, several histone modifications, and sites of DNA–protein interaction for several key regulatory factors. Widespread differences were identified in the composition and patterning of cytosine methylation between the two genomes. Nearly one-quarter of all methylation identified in embryonic stem cells was in a non-CG context, suggesting that embryonic stem cells may use different methylation mechanisms to affect gene regulation. Methylation in non-CG contexts showed enrichment in gene bodies and depletion in protein binding sites and enhancers. Non-CG methylation disappeared upon induced differentiation of the embryonic stem cells, and was restored in induced pluripotent stem cells. We identified hundreds of differentially methylated regions proximal to genes involved in pluripotency and differentiation, and widespread reduced methylation levels in fibroblasts associated with lower transcriptional activity. These reference epigenomes provide a foundation for future studies exploring this key epigenetic modification in human disease and development.

Highly Integrated Single-Base Resolution Maps of the Epigenome in Arabidopsis

Deciphering the multiple layers of epigenetic regulation that control transcription is critical to understanding how plants develop and respond to their environment. Using sequencing-by-synthesis technology we directly sequenced the cytosine methylome (methylC-seq), transcriptome (mRNA-seq), and small RNA transcriptome (smRNA-seq) to generate highly integrated epigenome maps for wild-type Arabidopsis thaliana and mutants defective in DNA methyltransferase or demethylase activity. At single-base resolution we discovered extensive, previously undetected DNA methylation, identified the context and level of methylation at each site, and observed local sequence effects upon methylation state. Deep sequencing of smRNAs revealed a direct relationship between the location of smRNAs and DNA methylation, perturbation of smRNA biogenesis upon loss of CpG DNA methylation, and a tendency for smRNAs to direct strand-specific DNA methylation in regions of RNA-DNA homology. Finally, strand-specific mRNA-seq revealed altered transcript abundance of hundreds of genes, transposons, and unannotated intergenic transcripts upon modification of the DNA methylation state.

Experimental Analysis of the Arabidopsis Mitochondrial Proteome Highlights Signaling and Regulatory Components, Provides Assessment of Targeting Prediction Programs, and Indicates Plant-Specific Mitochondrial Proteins

A novel insight into Arabidopsis mitochondrial function was revealed from a large experimental proteome derived by liquid chromatography–tandem mass spectrometry. Within the experimental set of 416 identified proteins, a significant number of low-abundance proteins involved in DNA synthesis, transcriptional regulation, protein complex assembly, and cellular signaling were discovered. Nearly 20% of the experimentally identified proteins are of unknown function, suggesting a wealth of undiscovered mitochondrial functions in plants. Only approximately half of the experimental set is predicted to be mitochondrial by targeting prediction programs, allowing an assessment of the benefits and limitations of these programs in determining plant mitochondrial proteomes. Maps of putative orthology networks between yeast, human, and Arabidopsis mitochondrial proteomes and the Rickettsia prowazekii proteome provide detailed insights into the divergence of the plant mitochondrial proteome from those of other eukaryotes. These show a clear set of putative cross-species orthologs in the core metabolic functions of mitochondria, whereas considerable diversity exists in many signaling and regulatory functions.

SUBA: the Arabidopsis Subcellular Database

Knowledge of protein localisation contributes towards our understanding of protein function and of biological inter-relationships. A variety of experimental methods are currently being used to produce localisation data that need to be made accessible in an integrated manner. Chimeric fluorescent fusion proteins have been used to define subcellular localisations with at least 1100 related experiments completed in Arabidopsis. More recently, many studies have employed mass spectrometry to undertake proteomic surveys of subcellular components in Arabidopsis yielding localisation information for approximately 2600 proteins. Further protein localisation information may be obtained from other literature references to analysis of locations (AmiGO: approximately 900 proteins), location information from Swiss-Prot annotations (approximately 2000 proteins); and location inferred from gene descriptions (approximately 2700 proteins). Additionally, an increasing volume of available software provides location prediction information for proteins based on amino acid sequence. We have undertaken to bring these various data sources together to build SUBA, a SUBcellular location database for Arabidopsis proteins. The localisation data in SUBA encompasses 10 distinct subcellular locations, >6743 non-redundant proteins and represents the proteins encoded in the transcripts responsible for 51% of Arabidopsis expressed sequence tags. The SUBA database provides a powerful means by which to assess protein subcellular localisation in Arabidopsis (http://www.suba.bcs.uwa.edu.au).

Organization and Regulation of Mitochondrial Respiration in Plants

Mitochondrial respiration in plants provides energy for biosynthesis, and its balance with photosynthesis determines the rate of plant biomass accumulation. We describe recent advances in our understanding of the mitochondrial respiratory machinery of cells, including the presence of a classical oxidative phosphorylation system linked to the cytosol by transporters, discussed alongside nonphosphorylating (and, therefore, non-energy conserving) bypasses that alter the efficiency of ATP synthesis and play a role in oxidative stress responses in plants. We consider respiratory regulation in the context of the contrasting roles mitochondria play in different tissues, from photosynthetic leaves to nutrient-acquiring roots. We focus on the molecular nature of this regulation at transcriptional and post-transcriptional levels that allow the respiratory apparatus of plants to help shape organ development and the response of plants to environmental stress. We highlight the challenges for future research considering spatial and temporal changes of respiration in response to changing climatic conditions.

Control of Ascorbate Synthesis by Respiration and Its Implications for Stress Responses

We show for the first time that respiration can control ascorbate (AA) synthesis in plants. Evidence for this control is provided by (a) the localization of l-galactono-1,4-lactone dehydrogenase (GalLDH), the terminal enzyme in AA biosynthesis, with mitochondrial complex I, and its regulation by

The Absence of ALTERNATIVE OXIDASE1a in Arabidopsis Results in Acute Sensitivity to Combined Light and Drought Stress

Treatment of Arabidopsis (Arabidopsis thaliana) alternative oxidase1a (aox1a) mutant plants with moderate light under drought conditions resulted in a phenotypic difference compared with ecotype Columbia (Col-0), as evidenced by a 10-fold increase in the accumulation of anthocyanins in leaves, alterations in photosynthetic efficiency, and increased superoxide radical and reduced root growth at the early stages of seedling growth. Analysis of metabolite profiles revealed significant changes upon treatment in aox1a plants typical of combined stress treatments, and these were less pronounced or absent in Col-0 plants. These changes were accompanied by alteration in the abundance of a variety of transcripts during the stress treatment, providing a molecular fingerprint for the stress-induced phenotype of aox1a plants. Transcripts encoding proteins involved in the synthesis of anthocyanins, transcription factors, chloroplastic and mitochondrial components, cell wall synthesis, and sucrose and starch metabolism changed, indicating that effects were not confined to mitochondria, where the AOX1a protein is located. Microarray and quantitative reverse transcription-polymerase chain reaction analysis revealed that transcripts typically induced upon stress treatment or involved in antioxidant defense systems, especially chloroplast-located antioxidant defense components, had altered basal levels in untreated aox1a plants, suggesting a significant change in the basal equilibrium of signaling pathways that regulate these components. Taken together, these results indicate that aox1a plants have a greatly altered stress response even when mitochondria or the mitochondrial electron transport chain are not the primary target of the stress and that AOX1a plays a broad role in determining the normal redox balance in the cell.

Organic acid activation of the alterNatlve oxidase of plant mitochondria

Alternative oxidase activity (oxygen uptake in the presence of KCN, antimycin or myxothiazol) in mitochondria isolated from the roots of soybean seedlings was very slow, even with succinate as substrate. This activity was stimulated substantially (100–400%) by the addition of pyruvate, with half maximal stimulation occurring at 0.1 mM pyruvate. Mitochondria from soybean shoots displayed high alternative oxidase activity with succinate and malate as substrates but lower activity with exogenous NADH; addition of pyruvate stimulated the activity with NADH up to that seen with succinate. This stimulation of cyanide‐insensitive NADH oxidation was seen also with mitochondria from other species. Hydroxypyruvate and oxoglutarate could substitute for pyruvate, although higher concentrations were required to achieve maximum stimulation. Pyruvate stimulation of cyanide‐insensitive oxygen uptake was observed with exogenous quinols as substrates, with sub‐mitochondrial particles, and in the presence of the pyruvate transport inhibitor, cyanohydroxycinnamic acid, but was not observed with detergent‐solubilised mitochondria. It is suggested that pyruvate acts allosterically on the alternatlve oxidase to stimulate its activity. The implications of these findings for respiration in vivo are discussed.

Stress-induced co-expression of alternative respiratory chain components in Arabidopsis thaliana

Plant mitochondria contain non-phosphorylating bypasses of the respiratory chain, catalysed by the alternative oxidase (AOX) and alternative NADH dehydrogenases (NDH), as well as uncoupling (UCP) protein. Each of these components either circumvents or short-circuits proton translocation pathways, and each is encoded by a small gene family in Arabidopsis. Whole genome microarray experiments were performed with suspension cell cultures to examine the effects of various 3 h treatments designed to induce abiotic stress. The expression of over 60 genes encoding components of the classical, phosphorylating respiratory chain and tricarboxylic acid cycle remained largely constant when cells were subjected to a broad range of abiotic stresses, but expression of the alternative components responded differentially to the various treatments. In detailed time-course quantitative PCR analysis, specific members of both AOX and NDH gene families displayed coordinated responses to treatments. In particular, the co-expression of AOX1a and NDB2 observed under a number of treatments suggested co-regulation that may be directed by common sequence elements arranged hierarchically in the upstream promoter regions of these genes. A series of treatment sets were identified, representing the response of specific AOX and NDH genes to mitochondrial inhibition, plastid inhibition and abiotic stresses. These treatment sets emphasise the multiplicity of pathways affecting alternative electron transport components in plants.

A Predicted Interactome for Arabidopsis

The complex cellular functions of an organism frequently rely on physical interactions between proteins. A map of all protein-protein interactions, an interactome, is thus an invaluable tool. We present an interactome for Arabidopsis (Arabidopsis thaliana) predicted from interacting orthologs in yeast (Saccharomyces cerevisiae), nematode worm (Caenorhabditis elegans), fruitfly (Drosophila melanogaster), and human (Homo sapiens). As an internal quality control, a confidence value was generated based on the amount of supporting evidence for each interaction. A total of 1,159 high confidence, 5,913 medium confidence, and 12,907 low confidence interactions were identified for 3,617 conserved Arabidopsis proteins. There was significant coexpression of genes whose proteins were predicted to interact, even among low confidence interactions. Interacting proteins were also significantly more likely to be found within the same subcellular location, and significantly less likely to be found in conflicting localizations than randomly paired proteins. A notable exception was that proteins located in the Golgi were more likely to interact with Golgi, vacuolar, or endoplasmic reticulum sorted proteins, indicating possible docking or trafficking interactions. These predictions can aid researchers by extending known complexes and pathways with candidate proteins. In addition we have predicted interactions for many previously unknown proteins in known pathways and complexes. We present this interactome, and an online Web interface the Arabidopsis Interactions Viewer, as a first step toward understanding global signaling in Arabidopsis, and to whet the appetite for those who are awaiting results from high-throughput experimental approaches.