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V7 Arabidopsis thaliana

V7 Arabidopsis thaliana. Arabidopsis thaliana is a small flowering plant that is widely used as a model organism in plant biology. Arabidopsis is a member of the mustard ( Brassicaceae ) family, which includes cultivated species such as cabbage and radish.

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V7 Arabidopsis thaliana

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  1. V7 Arabidopsis thaliana Arabidopsis thaliana is a small flowering plant that is widely used as a model organism in plant biology. Arabidopsis is a member of the mustard (Brassicaceae) family, which includes cultivated species such as cabbage and radish. Arabidopsis is not of major agronomic significance, but it offers important advantages for basic research in genetics and molecular biology. TAIR Biological Sequence Analysis

  2. Some useful statistics for Arabidopsis thaliana • Its small genome (114.5 Mb/125 Mb total) has been sequenced in the year 2000. • Extensive genetic and physical maps of all 5 chromosomes. • A rapid life cycle (about 6 weeks from germination to mature seed). • Prolific seed production and easy cultivation in restricted space. • Efficient transformation methods utilizing Agrobacterium tumefaciens. • A large number of mutant lines and genomic resources many of which are available from Stock Centers. • Multinational research community of academic, government and industry laboratories. TAIR Such advantages have made Arabidopsis a model organism for studies of the cellular and molecular biology of flowering plants. TAIR collects and makes available the information arising from these efforts. Biological Sequence Analysis

  3. Arabidopsis thaliana chromosomes red: Sequenced portions, light blue: telomeric and centromeric regions, black: heterochromatic knobs, magenta: rDNA repeat regions Gene density (`Genes') ranges from 38 per 100 kb to 1 gene per 100 kb; expressed sequence tag matches (`ESTs') ranges from more than 200 per 100 kb to 1 per 100 kb. Transposable element densities (`TEs') ranged from 33 per 100 kb to 1 per 100 kb. Black and green ticks marks: Mitochondrial and chloroplast insertions (`MT/CP'). black and red ticks marks: Transfer RNAs and small nucleolar RNAs (`RNAs') DAPI-stained chromophores Nature 408, 796 (2000) Biological Sequence Analysis

  4. Arabidopsis thaliana genome sequence The proportion of Arabidopsis proteins having related counterparts in eukaryotic genomes varies by a factor of 2 to 3 depending on the functional category. Only 8 ± 23% of Arabidopsis proteins involved in transcription have related genes in other eukaryotic genomes, reflecting the independent evolution of many plant transcription factors. In contrast, 48 ± 60% of genes involved in protein synthesis have counterparts in the other eukaryotic genomes, reflecting highly conserved gene functions. The relatively high proportion of matches between Arabidopsis and bacterial proteins in the categories `metabolism' and `energy' reflects both the acquisition of bacterial genes from the ancestor of the plastid and high conservation of sequences across all species. Finally, a comparison between unicellular and multicellular eukaryotes indicates that Arabidopsis genes involved in cellular communication and signal transduction have more counterparts in multicellular eukaryotes than in yeast, reflecting the need for sets of genes for communication in multicellular organisms. Nature 408, 796 (2000) Biological Sequence Analysis

  5. Many genes were duplicated Nature 408, 796 (2000) Biological Sequence Analysis

  6. Segmental duplication Segmentally duplicated regions in the Arabidopsis genome. Individual chromosomes are depicted as horizontal grey bars (with chromosome 1 at the top), centromeres are marked black. Coloured bands connect corresponding duplicated segments. Similarity between the rDNA repeats are excluded. Duplicated segments in reversed orientation are connected with twisted coloured bands. Nature 408, 796 (2000) Biological Sequence Analysis

  7. Membrane channels and transporters • Transporters in the plasma and intracellular membranes of Arabidopsis are responsible for the acquisition, redistribution and compartmentali-zation of organic nutrients and inorganic ions, for the efflux of toxic compounds and metabolic end products, energy and signal transduction. • almost half of the Arabidopsis channel proteins are aquaporins which emphasizes the importance of hydraulics in a wide range of plant processes. • - Compared with other sequenced organisms, Arabidopsis has 10-fold more predicted peptide transporters, primarily of the proton-dependent oligopeptide transport (POT) family, emphasizing the importance of peptide transport or indicating that there is broader substrate specificity than previously realized. • - nearly 1,000 Arabidopsis genes encoding Ser/Thr protein kinases, suggesting that peptides may have an important role in plant signalling. Nature 408, 796 (2000) Biological Sequence Analysis

  8. What is TAIR*? • NSF-funded project begun in 1999 • Web resource for Arabidopsis data and stocks • Literature-based manual annotation of gene function • Genome annotation (gene structure, computational gene function) URL The following slides were borrowed from a talk at the TAIR7 workshop by Eva Huala & Donghui Li * Biological Sequence Analysis

  9. Portals Biological Sequence Analysis

  10. Tools Biological Sequence Analysis

  11. Search Biological Sequence Analysis

  12. Biological Sequence Analysis

  13. Names Description Biological Sequence Analysis

  14. GO annotations Expression Biological Sequence Analysis

  15. Sequences Maps Biological Sequence Analysis

  16. Mutations Seed lines Biological Sequence Analysis

  17. Seed lines Links to other sites Biological Sequence Analysis

  18. Seed lines Links to other sites Biological Sequence Analysis

  19. Seed lines Links to other sites Biological Sequence Analysis

  20. Seed lines Links to other sites Biological Sequence Analysis

  21. Comments References Biological Sequence Analysis

  22. Biological Sequence Analysis

  23. Biological Sequence Analysis

  24. Biological Sequence Analysis

  25. Biological Sequence Analysis

  26. GBrowse - coming soon Biological Sequence Analysis

  27. Overview of releases to date 26,819 protein coding genes 3,866 alternatively spliced Biological Sequence Analysis

  28. Plant epigenetics - review • The genomes of several plants have been sequenced, and those of many others are under way. • But genetic information alone cannot fully address the fundamental question of • how genes are differentially expressed during cell differentiation and plant development, as the DNA sequences in all cells in a plant are essentially the • same. • Several important mechanisms regulate transcription by affecting the structural properties of the chromatin: • DNA cytosine methylation, • covalent modifications of histones, and • certain aspects of RNA interference (RNAi), • They are referred to as “epigenetic” because they direct “the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states”. Zhang, Science 320, 489 (2008) Biological Sequence Analysis

  29. The epigenetic landscape of A. thaliana diagram of chromosome. euchromatic arms, pericentromeric heterochromatin; centromeric core. The relative abundance of genes, repeats, cytosine methylation and siRNAs is shown for the length of A. thaliana chromosome 1, which is ~30 Mb long. Henderson & Jacobson, Nature 447, 418 (2007) Biological Sequence Analysis

  30. DNA methylation 3 distinct DNA methylation pathways with overlapping functions have been characterized in Arabidopsis. 1 The mammalian DNMT1 homolog METHYLTRANSFERASE 1 (MET1) primarily maintains DNA methylation at CG sites (CG methylation). 2 The plant-specific CHROMOMETHYLASE3 (CMT3) interacts with the H3 Lys9 dimethylation (H3K9me2) pathway to maintain DNA methylation at CHG sites (CHG methylation, H = A, C, or T). 3 The DNMT3a/3b homologs DOMAINS REARRANGED METHYLASE 1 and 2 (DRM1/2) maintain DNA methylation at CHH sites (CHH methylation), which requires the active targeting of small interfering RNAs (siRNAs). Zhang, Science 320, 489 (2008) Biological Sequence Analysis

  31. DNA methylation • Methylated and unmethylated DNA can be distinguished by 3 major types • of experimental approaches: • sodium bisulfite treatment that converts cytosine (but not methylcytosine) to uracil, • enzymatic digestion (using methylation-specific endonucleases or methylation sensitive isoschizomers), and • affinity purification or immunoprecipitation (with methyl-cytosine binding proteins and antibodies to methyl-cytosine, respectively). • The methylated fraction of the genome is then visualized by hybridizing treated • DNA to microarrays. Zhang, Science 320, 489 (2008) Biological Sequence Analysis

  32. DNA methylation Results from these microarray studies were largely consistent: 1 ~20% of the Arabidopsis genome is methylated. 2 Transposons and other repeats comprise the largest fraction of methlyated sequence. The promoters of endogenous genes are rarely methylated. 3 Surprisingly, methylation in the transcribed regions of endogenous genes is unexpectedly rampant (dt. ungezügelt). 4 More than one-third of all genes contain methylation (called “body methylation”) that is enriched in the 3′ half of the transcribed regions and primarily occurs at CG sites. Zhang, Science 320, 489 (2008) Biological Sequence Analysis

  33. DNA methylation DNA methylation is critically important in silencing transposons and regulating plant development. Severe loss of methylation results in a genome-wide massive transcriptional reactivation of transposons, and quadruple mutations in drm1 drm2 cmt3 met1 cause embryo lethality. Interestingly, the role of DNA methylation in regulating transcription appears to depend on the position of methylation relative to genes: - Methylation in promoters appears to repress transcription. - Paradoxically, however, body-methylated genes are usually transcribed at moderate to high levels and are transcribed less tissue-specifically relative to unmethylated genes. Zhang, Science 320, 489 (2008) Biological Sequence Analysis

  34. DNA methylation: new paper Recently, Cokus et al. combined sodium bisulfite treatment of genomic DNA with ultrahigh-throughput sequencing (>20× genome coverage) to generate the first DNA methylation map for any organism at single-base resolution. This “BS-Seq” method has several advantages over microarray-based methods : 1 it can detect methylation in important genomic regions that are not covered by any microarray platform (such as telomeres, ribosomal DNA, etc.). 2 it reveals the sequence contexts of DNA methylation (i.e., CG, CHG, and CHH) and therefore provides important information regarding the epigenetic pathways that function at any given locus. E.g. all 3 types of methylation colocalize to trans-posons, but gene body methylation occurs exclusively exclusively at CG sites. 3 BS-Seq is more effective in detecting light methylation and subtle changes (e.g., in mutants). 4 the theoretically unlimited sequencing depth makes it possible to quantitatively measure the percentage of cells in which any particular cytosine is methylated, thereby offering important clues regarding potential cell-specific DNA methylation. Biological Sequence Analysis

  35. RNA-directed DNA methylation Putative pathway for RNA directed DNA methylation in A. thaliana. Target loci (in this case tandemly repeated sequences; coloured arrows) recruit an RNA polymerase IV complex consisting of NRPD1A and NRPD2 through an unknown mechanism, and this results in the generation of a single-stranded RNA (ssRNA) species. This ssRNA is converted to double-stranded RNA (dsRNA) by the RNA-dependent RNA polymerase RDR2. The dsRNA is then processed into 24-nucleotide siRNAs by DCL3. The siRNAs are subsequently loaded into the protein AGO4, which associates with another form of the RNA polymerase IV complex, NRPD1B–NRPD2. AGO4 that is ‘programmed’ with siRNAs can then locate homologous genomic sequences and guide the protein DRM2, which has de novo cytosine methyltransferase activity. Targeting of DRM2 to DNA sequences also involves the chromatin remodelling protein DRD1. Henderson & Jacobson, Nature 447, 418 (2007) Biological Sequence Analysis

  36. DNA methyltransferase structure and function Plant and mammalian genomes encode homologous cytosine methyltransferases, of which there are 3 classes in plants and 2 in mammals. PWWP, Pro-Trp-Trp-Pro motif; UBA, ubiquitin associated. A. thaliana MET1 and Homo sapiens DNMT1 both function to maintain CG methylation after DNA replication, through a preference for hemi methylated substrates. Both have N-terminal BAH domains of unknown function. De novo DNA methylation is carried out by the homologous proteins DRM2 (in A. thaliana) and DNMT3A and DNMT3B (both in H. sapiens). Despite their homology, these proteins have distinct N-terminal domains, and the catalytic motifs present in the cytosine methyltransferase domain are ordered differently in DRM2 and the DNMT3 proteins. Plants also have another class of methyltransferase, which is not found in mammals. CMT3 functions together with DRM2 to maintain non-CG methylation. Henderson & Jacobson, Nature 447, 418 (2007) Biological Sequence Analysis

  37. Motiv density along Arabidopsis chromosomes Distribution of genes, repetitive sequences, DNA methylation, siRNAs, H3K27me3, and low nucleosome density (LND) regions. Left: chromosomal distributions on chr 1. The x axis shows chromosomal position. Zhang, Science 320, 489 (2008) Biological Sequence Analysis

  38. Small RNAs 4 major endogenous RNAi pathways have been described in Arabidopsis. Functioning at at the posttranscriptional level through mRNA degradation and/or translation inhibition are - the microRNA (miRNA), - transacting siRNA (ta-siRNA), and - natural-antisense siRNA (nat-siRNA) pathways. The siRNA pathway is involved in gene silencing both transcriptionally by directing DNA methylation and posttranscriptionally by guiding mRNA cleavage. Zhang, Science 320, 489 (2008) Biological Sequence Analysis

  39. Function of small RNAs MicroRNAs (miRNAs) and transacting siRNAs (tasiRNAs) are primarily involved in regulating gene expression and plant development, siRNAs play a major role in defending the genome against the proliferation of invading viruses and endogenous transposable elements. The function of the fourth type of sRNAs, natural-antisense siRNAs (nat-siRNAs), is not entirely clear but is likely related to plant stress responses Zhang et al., PNAS 104, 4536 (2007) Biological Sequence Analysis

  40. Small RNAs Millions of 21- to 24-nucleotide (nt) siRNAs have been cloned and sequenced from wild-type Arabidopsis plants and siRNA pathway mutants. Most of these studies generated not only sequence information necessary to map the siRNAs back to their originating genomic loci, but also the length information of siRNAs that is indicative of the processing enzymes involved (e.g., DICER-LIKE enzymes, DCLs). Zhang, Science 320, 489 (2008) Biological Sequence Analysis

  41. Small RNAs The majority of the siRNAs (>90%) are produced from double-stranded RNA (dsRNA) precursors generated by RNA polymerase IV isoform a (Pol IVa) and RNA-dependent RNA polymerase 2 (RDR2). RNAP IV is a recently identified class of RNAP that is specific to plant genomes. Unlike RNAP I, II, and III, RNAP IV appears to be specialized in siRNA metabolism. These dsRNA precursors are then processed by DCL3 to 24-nt siRNAs (with partially redundant contributions from DCL2 and DCL4) and become preferentially associated with ARGONAUTE4, which then interacts withPol IVb to direct DRM1/2- mediated CHH methylation. Most of these siRNAs are derived from genomic loci corresponding to transposons with high levels of CHH DNA methylation, and very few are found in protein-coding genes. Zhang, Science 320, 489 (2008) Biological Sequence Analysis

  42. Distribution patterns and transcription activity detailed distribution patterns and transcription activity (vertical blue bars) in a gene-rich region (top) and a repeat-rich region (bottom). Red boxes: genes; Arrows indicate the direction of transcription. Zhang, Science 320, 489 (2008) Biological Sequence Analysis

  43. Positioning relative to Arabidopsis genes (A) Distribution of DNA methylation, siRNAs, and H3K27me3 relative to Arabidopsis genes. Thick and thin horizontal bars represent genes and intergenic regions, respectively. (B) Distribution of repetitive sequences relative to genes in Arabidopsis (green) and rice (red). Zhang, Science 320, 489 (2008) Biological Sequence Analysis

  44. Conclusions 2 major fractions of the Arabidopsis genome are associated with and regulated by different epigenetic mechanisms: (1) Genes are regulated by pathways such as H3K27me3, H3K4me2, and miRNAs/ta-siRNAs/nat-siRNAs, whereas (2) transposons and other repeats are silenced by DNA methylation, H3K9me2, and siRNAs. Such a functional distinction, however, is blurred when the 2 genetic fractions overlap, which occurs much more frequently in larger and more complex genomes. Zhang, Science 320, 489 (2008) Biological Sequence Analysis

  45. Conclusions Although increasingly comprehensive, such an epigenomic picture remains static. Relatively little is known about how the plant epigenome changes in response to developmental or environmental cues. A particularly interesting question may be how mechanisms that evolved to stably silence transposons could offer the flexibility required for the developmental regulation of endogenous genes. In addition, we do not yet have a clear understanding of the nature and the maintenance of the boundaries separating epigenetically distinct chromatin compartments. In some cases, genetic landmarks (such as the transcription unit) may serve as borders; in other cases, the balancing acts of opposing epigenetic mechanisms may help to stably maintain the epigenetic landscape of plant genomes. Zhang, Science 320, 489 (2008) Biological Sequence Analysis

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