1. Organelle genomes Organelle gene expression processes Organelle-to-nucleus signaling (retrograde regulation) PCB6528 Plant Cell and Developmental Biology Spring 2013 Organelle genomes, gene expression and signaling Christine Chase – 2215 Fifield Hall – 352-273-4862 cdchase@ufl.edu

2. Describe the organization and coding content of plant plastid and mitochondrial genomes Discuss the similarities and differences between the plastid and plant mitochondrial genomes with respect to organization and evolution Explain why organelle coding content is not identical between plant species Discuss the possible reasons that plant organelles retain genomes at all Describe the process of plastid genome transformation Discuss the utility and applications of plastid transformation and provide some specific examples Objectives - Organelle genomes:

3. Organelle genome databases: http://www.hsls.pitt.edu/obrc/index.php?page=or ganelle Small but essential Multiple organelles per cell, multiple genomes per organelle 20 – 20,000 genomes per cell depending on cell type Organized in nucleo-protein complexes called nucleoids Non-Mendelian inheritance usually but not always maternal Necessary but insufficient to elaborate a functional organelle nuclear gene products required translated on cytosolic ribosomes imported into the organelles plant mitochondria also import tRNAs Organelle genomes

4. Comparative sizes of plant genomes GenomeSize in bp Arabidopsis thaliana nuclear 1.4 x 10 8 Arabidopsis thaliana mitochondria 3.7 x 10 5 Arabidopsis thaliana plastid 1.5 x 10 5 Zea mays nuclear 2.4 x 10 9 Zea mays mitochondria 5.7 x 10 5 Zea mays plastid 1.4 x 10 5

5. Target P prediction analysis of the complete Arabidopsis nuclear genome sequence (Emanuelsson et al., J Mol Biol 300:1005)says..... ~ 10% of the Arabidopsis nuclear genome (~2,500 genes) encode proteins targeted to the mitochondria ~ 14% of the Arabidopsis nuclear genome (~3,500 genes) encodes proteins targeted to the plastid So 25% of the Arabidopsis nuclear genome is dedicated to organelle function! Proteome reflects metabolic diversity of these organelles, both anabolic and catabolic Organelle genomics & proteomics

6. * * * [Gillham 1994 Organelle Genes & Genomes] Endosymbiont origin of organelles Original basis in cytology Confirmation by molecular biology α proteobacteria as closest living relatives to mitochondria Cyanobacteria closest living relatives to plastids Archaebacteria considered to be related to primitive donor of the nuclear genome * * *

7. Chimeric origin of eukaryotic nuclear genomes Genes per category among 383 eubacterial- & 111 archeaebacterial- related genes in the yeast nuclear genome Esser et al. 2004 Mol Biol & Evol 21:1643

8. Evolution of mitochondrial genome coding content GenomeProtein coding genes Rikettsia prowazekii (smallest  proteobacterial genome) 832 Reclinomonas americana mitochondria (protozoan; most mitochondrial genes) 62 Marchantia polymorpha mitochondria 1.9 x 10 5 bp (liverwort, non-vascular plant ) 64 Arabidopsis thaliana mitochondria 3.7 x 10 5 bp (vascular plant) 57 Homo sapiens mitochondria 13

9. Evolution of plastid genome coding content GenomeProtein coding genes Synechococcus (cyanobacteria) 3,300 Paulinella chromatophora photosynthetic body (endosymbiont cyanobacteria) 867 Porphyra purpurea plastid (red alga) 209 Chlamydomonas reinhardtii plastid (green alga) 63 Marchantia polymorpha plastid (liverwort, non-vascular plant) 67 Arabidopsis thaliana plastid (vascular plant) 71 Epifagus virginiana plastid (non-photosynthetic parasitic plant) 42

10. Evolution of the eukaryotic genomes Reduced coding content of organelle genomes compared to endosymbiont Functional gene transfer to nucleus with protein targeted back to organelle Functional re-shuffling - organelles replace prokaryotic features with eukaryotic, “hybrid” or novel features

11. Functional gene transfer from organelle to nuclear genome Gene by gene Evidence for frequent and recent transfers in plant lineage Results in coding content differences among plant organelle genomes What is required for a functional gene re-location from organelle to nucleus?

12. Southern blot hybridization of total cellular DNA Mitochondrial nad1 and rps10 probes Shading = taxa with no hybridization to rps10 Bullets = taxa with confirmed nuclear rps10 gene Why no hybridization of rps10 probes to DNA with confirmed nuclear copy? (Hint: How are the relative genome copy numbers exploited in this screen?) What is the purpose of the nad1 probe? What are the implications of these findings for plant mitochondrial genome coding content? [Adams et al. Nature 408:354] Functional gene transfer: Recent repeated transfers of the plant mitochondrial rps10 to the nucleus

13. Non-Functional DNA transfer from organelle to nuclear genome Frequent Continual (can detect in “real-time” as well as evolutionary time) In large pieces e.g. Arabidopsis 262 kb numtDNA (nuclear-localized mitochondrial DNA) 88,000 years ago e.g. Rice 131 kb nupDNA (nuclear-localized plastid DNA) 148,000 years ago

14. Land Plant Plastid Genome Organization 120-160 kb depending on species conserved coding conserved physical organization Physical map restriction map or DNA sequence 120-160 kb circular genome Large inverted repeat (LIR) commonly 20-30 kb large single copy (LSC) region small single copy (SSC) region Active recombination within the LIR Expansion and contraction of LIR primary length polymorphism among land plant species 10-76 kb Some conifers and legumes have very reduced or no LIR SC region inversion polymorphisms mediated by infrequent recombination between small dispersed repeats

15. (Maier et al. J Mol Biol 251:614) Plastid genome organization

16. Plastid ATP synthase genes in operons (from Palmer [1991] in Cell Culture and Somatic Cell Genetics of Plants, V 7A. L Bogorad and IK Vasil eds. Academic Press, NY, pp 5-142)

17. The plastid genome oversimplified: recombination across inverted repeats leads to inversions How can these inversion isomers be detected? trn N rps19 rps15 psbA ndhF ndhB trn N ndhB rps19 rpl22 trn N rps19 rps15 psbA ndhF ndhB trn N ndhB rps19 rpl22

18. [Lilly et al. Plant Cell. 13:245] Fiber FISH of tobacco plastid DNA IR probe SSC+IR probe SC gene probes

19. Structural complexity of plastid DNA from tobacco, arabidopsis, and pea [Lilly et al. Plant Cell. 13:245] IR probe SSC+IR probe

20. [Lilly et al. Plant Cell. 13:245] Structural complexity of plastid DNA from tobacco, arabidopsis, and pea

21. Land plant mitochondrial genome organization 208-2400 kb depending on species Relatively constant coding but highly variable organization among and even within a species Physical mapping with overlapping cosmid clones Entire complexity maps as a single “master circle” All angiosperms except Brassica hirta have one or more recombination repeats Repeats not conserved among species Direct and/or inverted orientations on the “master” Recombination generated inversions (inverted repeats) Recombination generated subgenomic molecules (deletions) (direct repeats), some present at very low copy number (sublimons) Leads to complex multipartite structures

22. Recombination across direct repeats leads to deletions (subgenomic molecules) a’ b’ c d Pac I PmeI a b c d b’ c’ d’ a’ Pac I AscI a b c’ d’ Not I AscI How can these deletion (subgenomic) isomers be detected? a’ b’ c’ d’ a b c d Pac I AscI PmeI Not I b’ c’ d’ a’

23. Arabidopsis mitochondrial genome organization > > > > > > > > > > > > > > > > [modified from Backert et al. Trends Plant Sci 2:478] Two pairs of repeats active in recombination One direct (orange, top left) One inverted (blue, top left) Recombining the inverted (blue pair) creates an inversion What has happened to the orientation of the orange repeats (top right)?

24. (Backert and Börner, Curr Genet 37:304) Branched rosette and linear molecules from C. album mitochondria

25. [Backert et al. Trends Plant Sci 2:478] Structural complexity of plant mitochondrial DNA

26. Structural complexity of plant organelle genomes Plastid genomes map as a single circle Inversion isomers Indicate recombination through the LIR Plant mitochondrial genomes map as a single master circle plus Many subgenomic circles Inversion isomers Imply recombination through multiple direct & inverted repeat pairs Direct visualization via EM or FISH Rosette/knotted/branched structures Longer-than genome linear molecules Shorter-than genome linear and circular molecules Sigma molecules Branched linear molecules Few if any genome-length circular molecules (mitochondria only)

27. Circular maps from linear molecules fixed terminal redundancy (e.g. phage T7) ABCDEF______________XYZABC circularly permuted monomers ABCDEF______________XYZ BCDEF______________XYZA CDEF _____________ XYZAB circularly permuted monomers & terminal redundancy (e.g. phage T4) CDEF______________XYZABCDEF DEFG____________ XYZABCDEFG EFGH___________XYZABCDEFGH linear dimers or higher multimers ABCDEF__________XYZABCDEF_________XYZ A Z B Y C X D In a circular molecule or map, fragment A is linked to B, B to C, C to D, D to X, X to Y, Y to Z and Z to A. But these linkages also hold true for linear molecules

28. [Freifelder, 1983, Molecular Biology] Physical structures of DNA obtained via rolling circle DNA replication

29. Recombination dependent DNA replication [RDR] [Marechal and Brisson New Phytol 186:299]

30. Complex rosette/knotted structures nucleoids Longer-than genome linear molecules rolling circle replication intermolecular recombination of linear molecules Shorter-than genome linear and circular molecules intramolecular recombination between direct repeats Sigma molecules rolling circles recombination of circular & linear molecules Branched linear molecules recombination-mediated replication Few genome-length circular molecules (none for mitochondrial) What governs the stable inheritance of this mess? Origins of plant organelle genome complexity

31. Repair of DNA damage organelles rich in damaging ROS low rates of synonymous-substitution homologous recombination with gene conversion repair point mutations repair DNA breaks lots of wild-type recombination partners Genome replication structures support the recombination dependent replication model ? Does recombination also create a cohesive unit of inheritance Recombination and plant organelle genome stability

32. Recombination surveillance Restricts recombination between short repeats (~100-500 bp) in plant organelle DNAs Mediated by four protein families members targeted to plastids &/or mitochondria MSH1 - E. coli mismatch repair homologs RECA - Recombinase/homology search/strand invasion OSB - organelle single-stranded DNA binding proteins Whirly - single-stranded DNA binding proteins Recombination and plant organelle genome (in) stability

33. Plant organelle recombination surveillance team [Marechal and Brisson New Phytol 186:299]

34. Down-regulation of MSH1 alters organelle function and genome organization Mitochondrial genome reorganization left, co-segregating with and leaf variegation, right Organelle recombination is regulated De-regulation destabilizes organelle genome organization with phenotypic consequences Some recombination is good; too much is bad! [Sandhu et al. Proc Natl Acad Sci USA 104:1766

35. Plastid genome coding content Chloroplast Genome Database: http://chloroplast.cbio.psu.edu/ (Cui et al., Nucl Acids Res 34: D692-696) Generally conserved among land plants, more variable among algae Genes for plastid gene expression rRNAs, tRNAs ribosomal proteins RNA polymerase Genes involved in photosynthesis 28 thylakoid proteins Photosystem I (psa) Photosystem II (psb) ATP synthase subunits (atp) NADH dehydrogenase subunits (nad) Cytochrome b6f subunits (pet) RUBISCO large subunit (rbcL) (rbcS is nuclear encoded)

36. Plastid genomes encode integral membrane components of the photosynthetic complexes Photosynthetic composition of the thylakoid membrane Green = plastid-encoded subunits Red = nuclear-encoded subunits What do you notice about the plastid vs nuclear- encoded subunits ? What hypotheses does this suggest regarding the reasons for a plastid genome? [Leister, Trends Genet 19:47]

37. Plant mitochondrial genome coding content In organello protein synthesis estimates 30-50 proteins encoded by plant mitochondrial genomes Complete sequence of A. thaliana mit genome 57 genes respiratory complex components rRNAs, tRNAs, ribosomal proteins cytochrome c biogenesis Plant mit genomes lack a complete set of tRNAs mit encoded tRNAs of mit origin mit encoded tRNAs functional transfer from the plastid genome nuclear encoded tRNAs imported into mitochondria to complete the set 42 orfs that might be genes Gene density (1 gene per 8 kb) lower than the nuclear gene density (1 gene per 4-5 kb)!

38. Plant mitochondrial genome coding content Table 3 General features of mtDNA of angiosperms FeatureNta a AthBnaBvuOsa MC (bp)430,597366,924221,853368,799490,520 A+T content (%)55.055.254.856.156.2 Long repeated (bp) b 34,53211,3722,42732,489127,600 Unique c 39,206 37,549 38,065 34,499 40,065 Coding d (9.9%) (10.6%) (17.3%) (10.3%) (11.1%) Cis-splicing introns 25,617 28,312 28,332 18,727 26,238 (6.5%) (8.0%) (12.9%) (5.6%) (7.2%) ORFs e 46,773 37,071 20,085 54,288 12,009 (11.8%) (10.4%) (9.2%) (16.1%) (3.3%) cp-derived (bp)9,942 3,958 7,950 g 22,593 (2.5%) (1.1%) (3.6%) 2.1% h (6.2%) Others274,527 248,662 124,994 262,015 (69.3%) (69.9%) (57%) 65.9% (72.2%) Gene content f 6055535256 (from Sugiyama et al. Mol Gen Gen 272:603)

39. Mitochondrial genomes encode integral membrane components of the respiratory complexes = one mitochondria-encoded subunit * II AOX intermembrane space inner membrane matrix I UQH 2 UQ H+H+ CYC IV H+H+ III H+H+ H+H+ ATP Synthase II TCA cycle NADH NAD+ NAD(P)H DH external NAD(P)H DH internal 2H 2 O O2O2 O2O2 ADP ATP *** * **** *** * * There is some species-to-species variation with respect to the presence or absence of genes encoding respiratory chain subunits. What is the likely explanation for this observation? (Modified from Rasmusson et al. Annu Rev Plant Biol 55:23)

40. Plastid genome transformation DNA delivery by particle bombardment or PEG precipitation DNA incorporation by homologous recombination Initial transformants are heteroplasmic, having a mixture of transformed and non-transformed plastids Selection for resistance to spectinomycin (spec) and streptomycin (strep) antibiotics that inhibit plastid protein synthesis Spec or strep resistance conferred by individual 16S rRNA mutations Spec and strep resistance conferred by aadA gene (aminoglycoside adenylyl transferase) Untransformed callus bleached; transformed callus greens and can be regenerated Multiple selection cycles may be required to obtain homoplasmy (all plastid genomes of the same type)

41. Plastid genome transformation [Bock & Khan, Trends Biotechnol 22:311]

42. Selection for plastid transformants [Bock, J Mol Biol 312:425] A) leaf segments post bombardment with the aadA gene B) leaf segments after selection on spectinomycin C) transfer of transformants to spectinomycin + streptomycin D) recovery of homoplasmic spec + strep resistant transformants

43. Applications of plastid genome transformation by homologous recombination [Bock, Curr Opin Biotechnol 18:100]

44. [Hager et al. EMBO J 18:5834] Functional analysis of plastid ycf6 in transgenic plastids

45. Functional analysis of plastid ycf6 in transgenic plastids [Hager et al. EMBO J 18:5834] ycf6 knock-out lines: Homoplasmic for aadA insertion into ycf6 Pale-yellow phenotype Normal PSI function and subunit accumulation Normal PSII function and subunit accumulation Abnormal b6f (PET) subunit accumulation Mass spectrometry demonstrates YCF6 in normal plastid PET complex Why, if ycf6 is the disrupted gene, does another PET complex subunit (PETA) fail to accumulate ?

46. Non-functional plastid-to-nucleus DNA transfer Transform plastids with: plastid promoter – aadA linked to nuclear promoter - neo Pollinate wild-type plants with transformants % seed germination on kanamycin ~ frequency of nuclear promoter - neo transferred from plastid to nucleus Why does this experiment primarily estimate the frequency of DNA transfer from plastid to nucleus, rather than the frequency of functional gene transfer from plastid to nucleus? How would you re-design the experiment to test for features of a functional gene transfer? [Timmis et al. Nat Rev Genet 5:123]