PCB6528 Plant Cell and Developmental Biology Spring 2015

PCB6528 Plant Cell and Developmental Biology Spring 2015
Organelle genomes, gene expression and signaling Christine Chase – 2215 Fifield Hall –  Organelle genomes  Organelle gene expression  Organelle signaling

How do we build and maintain these highly abundant and complex respiratory and photosynthetic factories? Top: Composition of the photosynthetic apparatus in the thylakoid membrane, and summary of the mutants available. Known structural data were used to illustrate the composition of multiprotein complexes, but the positions of subunits within the complexes are largely arbitrary. Plastome-encoded subunits are indicated in light green, or dark green if inactivation of the corresponding gene has been reported. Nucleus-encoded proteins are depicted in orange, or red if knockout or downregulation of the protein has been achieved. Note that although mutants from both Arabidopsis and Chlamydomonas are considered, the composition of the photosynthetic apparatus refers only to Arabidopsis, which in this regard differs in some respects from Chlamydomonas. YCF3 and YCF4 (not shown) are localized on the thylakoid membrane, but not stably associated with a multiprotein complex. PC, plastocyanin; Fd, ferredoxin; FNR, ferredoxin-NADPH reductase; LHC, light-harvesting complex; PS, photosystem. Bottom: Schematic representation showing the proteins identified by GeLC-MS/MS related to the functionality of mitochondrial respiratory chain complexes. AOX, Alternative oxidase; ATPase, ATP synthase; CI to CV, respiratory chain complexes I to V; Cyt B, cytochrome b; Cyt C, cytochrome c; Cyt c1, cytochrome c1; COX, cytochrome oxidase; Ddh, dihydroorotate dehydrogenase; ETF, electron transfer flavoprotein; exNDH, external NADH-ubiquinone oxidoreductase; GLDH, l-galactono-1,4-lactone dehydrogenase; inNDH, internal NADH-ubiquinone oxidoreductase; NAD-DH, NADH dehydrogenase (ubiquinone) subcomplex; Succ DH, succinate dehydrogenase; UCP, uncoupling protein; UQox, ubiquinone oxidized; UQred, ubiquinone reduced. [Leister, Trends Genet 19:47; Salvato, Plant Physiol 164:637]

Objectives - Organelle genomes:
Describe the physical organization and coding content of modern-day organelle genomes Explain how we can reconcile discrepancies between physical maps and the observed DNA structures Describe how evolution has shaped and changed modern-day organelle genomes from their ancestral prokaryotic genomes Discuss the possible reasons that plant organelles retain genomes at all Discuss the challenges associated with genetic transformation of organelle genomes and how these challenges have been met to achieve genetic transformation of the plastid genome Discuss the utility and applications of plastid transformation and provide some specific examples

Organelle genome databases:
Organelle genomes Organelle genome databases: 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 not sufficient to elaborate a functional organelle nuclear gene products required translated on cytosolic ribosomes imported into the organelles plant mitochondria also import tRNAs

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

Organelle genomics & proteomics
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 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 * * [Gillham 1994 Organelle Genes & Genomes]

Eukaryotic nuclear genomes – a big mix & match experiment
383 eubacterial- & 111 archeaebacterial- related genes in the yeast nuclear genome Genes per category Compared 6214 yeast protein coding nuclear genes to 177,177 proteins predicted by sequenced eubacterial and archaebacterial genomes 850 FASTA matches with E-value of 10-20; 383 eubacterial; 111 archaebacterial; 263 widespread Note predominant archae origin of transcription & translation functions vs eubacterial origin of metabolic function The left portion of figure 3 shows the 50 most highly conserved yeast proteins that are specific to eubacterial and archaebacteria, respectively. Among the archaebacterial-specific genes several ribosomal proteins, DNA metabolic enzymes, and proteasome subunits are prominent. Core carbon metabolic, core biosynthetic, and glycolytic enzymes are prominent among the eubacterial genes. The latter finding is of interest because it has been claimed that eukaryotes do not possess eubacterial glycolytic enzymes (Canback, Andersson, and Kurland 2002). However, in the present taxon sample there are numerous glycolytic enzymes and other enzymes of core carbon metabolism among the 383 genes that do not occur in 15 sequenced archaebacterial genomes, including glyceraldehyde 3-phosphate dehydrogenase, triosephosphate isomerase, phosphoglycerate mutase (2,3-BPG-dependent), fructose-1,6-bisphosphatase, phosphoglucomutase, fructose-bisphosphate aldolase, glucose-6-phosphate isomerase, glucose-6-phosphate 1-dehydrogenase, phosphoenolpyruvate carboxykinase (ATP-dependent), NAD-dependent malic enzyme, glycerol-3-phosphate dehydrogenase, malate dehydrogenase, ribulose-phosphate 3-epimerase, transketolase, transaldolase, pyruvate decarboxylase, glycerol kinase, malate dehydrogenase, and invertase. (Esser et al Mol Biol & Evol 21:1643) Esser et al Mol Biol & Evol 21:1643

Evolution of mitochondrial genome coding content
Protein 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 bp (vascular plant) 57 Homo sapiens mitochondria 13

Evolution of plastid genome coding content
Protein 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

Evolution of the eukaryotic genomes
Reduced coding content of organelle genomes compared to endosymbiont Modern day organelle genomes don’t encode all the proteins required for organelle function Functional gene transfer to nucleus with protein targeted back to organelle Functional re-shuffling - organelles replace prokaryotic features with eukaryotic, “hybrid” or novel features

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?

Functional gene transfer: Recent repeated transfers of the plant mitochondrial rps10 to the nucleus
Figure 1: Southern blot hybridizations of 51 angiosperms (out of 277 examined in total) with the indicated angiosperm probes. Shading indicates taxa with no hybridization to the rps10 exon probe. Triangles indicate no hybridization to only the rps10 intron probe (a total of six intron losses were inferred); asterisk indicates no hybridization to only the exon probe. Bullets indicate species from which at least part of rps10 was isolated from the nucleus. [from Adams et al. Nature 408:354] 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]

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

Land Plant Plastid Genome Organization
kb depending on species conserved coding conserved physical organization Physical map restriction map or DNA sequence kb circular genome Large inverted repeat (LIR) commonly 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

Plastid genome organization
(Maier et al. J Mol Biol 251:614) Figure 1. A, Gene organization of the Zea mays plastome. The inverted repeat regions IRA and IRB, respectively, divide the rest of the circular genome into large (LSC) and small (SSC) single copy regions. Genes drawn outside the circle are transcribed clockwise. Genes in which editing sites have been detected are marked by framing. The numbers within the parentheses behind the gene symbols indicate the numbers of editing sites observed in the respective genes. The orientation of the entire SSCregion has been reversed as compared with the earlier version based on restriction site analysis (Larrinua et al., 1983). This reorientiation, representing one of the two possible structural isomers of a cp DNA molecule carrying IR-regions (Palmer, 1983), is now in accordance with the genetic map of the plastomes from tobacco, Marchantia polymorpha, rice and black pine, respectively. B, Length comparison of the completely sequenced higher plant plastomes. LSCs are marked by striations, SSCs by open boxes and IRs by filled boxes. Tobacco: Shinozaki et al. (1986); rice: Hiratsuka et al. (1989); Marchantia polymorpha (liverwort): Ohyama et al. (1986); black pine: Wakasugi et al. (1994); Epifagus: Wolfe et al. (1992). (Maier et al. J Mol Biol 251:614)

Plastid ATP synthase genes in operons
Note we do not see this for plant mitochondrial genomes, where gene order is highly scrambled! (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)

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

Fiber FISH of tobacco plastid DNA
IR probe SSC+IR probe SC gene probes Figure 2. Fiber-FISH Images of Tobacco Chloroplast DNA. (A) A monomeric circular cpDNA molecule. The green signal represents the entire cpDNA genome, and the red signal represents the 12.2-kb IR. (B) A monomeric circular cpDNA molecule with 87 kb (24 µm) of the green signal representing the LSC and 68 kb (17 µm) of the red signal homologous to both the SSC and IR regions. (C) to (E) Three monomeric cpDNA molecules (green) together with hybridization signals (red) derived from the (C), 4.6- (D), and 1.8-kb (E) cpDNA probes. (F) to (J) Schematic illustrations of the cpDNA images, corresponding to (A) to (E), respectively. Green and red lines represent the differential hybridization. Bars = 5 µm. [Lilly et al. Plant Cell. 13:245]

Structural complexity of plastid DNA from tobacco, arabidopsis, and pea
IR probe SSC+IR probe Figure 3. Figure 3. Structural Plasticity of cpDNA Molecules from Tobacco, Arabidopsis, and Pea. (A) A wide-field view (magnification x400) showing three circular cpDNA molecules from tobacco. The two small circles are monomer size, and the larger molecule is trimer size (arrow). (B) A field of view (magnification x630) showing tobacco chloroplast fibers hybridized with an IR probe (red FISH signal). (C) Selected monomeric and multimeric cpDNA molecules from tobacco. Red signals are from the 12.2-kb IR probe. (D) A complete linear hexameric tobacco molecule. The green signals represent LSC; the red signals show SSC and IR regions. (E) A circular tetrameric chloroplast molecule from Arabidopsis. The green signals were derived from P1 clones MAB17 and MCI3, and the red signals are from P1 clone MAH2 (Sato et al ). (F) Two monomeric circular molecules from pea probed with a single-copy sequence possessing the origin of replication (red). Bars = 10 µm. [Lilly et al. Plant Cell. 13:245]

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

Land plant mitochondrial genome organization
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

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

Arabidopsis mitochondrial genome organization
Fig. 1. Circular and linear models of the mtDNA structure in plants. The dynamics of the mtDNA of plants allow several models. a) A circular genome model representing the different organisations of the mtDNA of A. thaliana ecotype C24. The two pairs of large, frequently recombining repeats (A and B) and their orientations are represented by blue and red arrows. The different parts of the genome are colored in a range of gray scale. Intramolecular recombination events are represented with dashed lines, and in orange, recombination leading to subgenomic molecules. Dotted line shows possible intermolecular recombination. b) The mtDNA has been observed as predominantly constituted by a complex array of linear molecules. Branched forms of the mtDNA could be intermediates of recombination-dependent processes: 3′ single-stranded sequences resulting from the recession of double-strand breaks can invade homologous double-stranded DNA, forming a D-loop and leading to the establishment of replication forks. a) Two pairs of repeats active in recombination One direct (A, top left) One inverted (B, top left) Recombining the inverted pair creates an inversion What happens to the B pair ? Recombining the direct pair creates a deletion (2 subgenomes) that can further recombine b) Although physical mapping yields the organizations shown in a, optical mapping shows a very different organization [Gualberto et al. Biochimie 100:107]

Branched rosette and linear molecules from C. album mitochondria
Fig. 4a±d Detection of branched regions in rosette-like DNA molecules from C. album mitochondria. Four spread rosette structures having internal forks (small arrowheads) and/or linear DNA fibres with a loop at the end (large arrowheads) are shown (a±d). The arrow in b indicates a large stretch of collapsed single-stranded DNA (bar represents 1 kb) (Backert and Börner, Curr Genet. 37:304) (Backert and Börner, Curr Genet 37:304)

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

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 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 for mitochondria

Circular maps from linear molecules
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 A Z B Y C X D 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

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

Circular maps from linear molecules
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 A Z B Y C X D 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

Recombination dependent DNA replication
[RDR] Figure 1. Mechanisms of homologous recombination. (b) Single-ended DSBs produced by the demise of replication forks can be successfully repaired by the break-induced replication/recombination-dependent replication pathway (BIR/RDR). In the illustrated case, a single-stranded break on the lagging strand template leads to replication fork collapse. Then, as in DSBR and SDSA, resection of the 5′-end on the broken DNA frees a 3′-OH single-stranded overhang which can invade a homologous donor duplex. DNA synthesis starts and the extended 3′-end is unable to find a complementary second end. To synthesize the missing strand, a replication fork is established and polymerization proceeds to the end of the donor molecule. This results in gene conversion accompanied by nonreciprocal crossing-over. This figure is modified from Hastings et al. (2009b). [Marechal and Brisson New Phytol 186:299]

Origins of plant organelle genome complexity
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?

Recombination and plant organelle genome stability
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 (in) stability
Recombination surveillance Limits recombination between short repeats (~ bp) in plant organelle DNAs Mediated by four protein families members targeted to plastids &/or mitochondria MSH1 - Eubacterial mismatch repair homologs RECA - Eubacterial recombination homology search/strand invasion OSB - Plant-specific organelle single- stranded DNA binding proteins Whirly - Primarily plant, single- stranded DNA binding proteins

Plant organelle recombination surveillance team
Figure 3. Protein families of the organelle recombination surveillance machinery. (a) Rectangular representation of proteins with established roles in preventing aberrant recombination of plant organelle genomes. Arabidopsis MutS homologue 1 (MSH1) is a 120-kDa MutS homologue fused with a GIY-YIG endonuclease domain. Organellar RecA homologues are 40–45-kDa proteins containing a well-conserved bacteria-like RecA-fold. Organellar single-stranded DNA (ssDNA)-binding proteins (OSBs) are 30–50-kDa proteins which harbour an oligonucleotide/oligosaccharide binding (OB)-fold-like domain putatively involved in oligomerization and a variable number of ssDNA-binding PDF domains in their C-terminal region. Depicted here is the Arabidopsis OSB1 protein with a single PDF domain. Whirlies are c. 24-kDa proteins that bind single-stranded DNA and RNA through their highly-conserved central Whirly domain. TP, transit peptide. (b) Crystallographic three-dimensional structure of Solanum tuberosum Whirly 1 (StWhy1). Left, cartoon representation of an StWhy1 protomer with β-strands coloured in yellow, α-helices in red and loops in green. Right, cartoon representation of an StWhy1 tetramer showing its whirligig-like appearance (adapted fromDesveaux et al., 2002). These representations were generated using PyMOL (DeLano, 2002). [Marechal and Brisson New Phytol 186:299]

Organelle recombination is regulated
Down-regulation of MSH1 alters organelle function and genome organization Mitochondrial genome reorganization left, co-segregating with leaf variegation, right Organelle recombination is regulated De-regulation destabilizes organelle genome organization with phenotypic consequences Some recombination is good; too much is bad! Fig. 3.The male sterility phenotype observed in transgenic tomato lines. (A) DNA gel blot hybridization analysis of total genomic DNA from Rutgers (wt) and Rutgers male sterile (ms) T0 transgenic line 17 digested with PstI/SalI. The mitochondrial DNA probe, derived from A. thaliana BAC clone T5E7, encompasses eight mitochondrial genes and/or ORFs. Evidence of DNA polymorphism in the male sterile line is shown by an arrowhead. (B) Some leaves from a transgenic Rutgers T1 semisterile plant display a green-white variegation pattern that appears to be associated with the mitochondrial DNA rearrangement shown inA. [Sandhu et al. Proc Natl Acad Sci USA 104:1766]

Plastid genome coding content
Chloroplast Genome Database: (Cui et al., Nucl Acids Res 34: D ) 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)

Plastid genomes encode integral membrane components of the photosynthetic complexes
Fig. 2. Composition of the photosynthetic apparatus in the thylakoid membrane, and summary of the mutants available. Known structural data were used to illustrate the composition of multiprotein complexes, but the positions of subunits within the complexes are largely arbitrary. Plastome-encoded subunits are indicated in light green, or dark green if inactivation of the corresponding gene has been reported. Nucleus-encoded proteins are depicted in orange, or red if knockout or downregulation of the protein has been achieved. Note that although mutants from both Arabidopsis and Chlamydomonas are considered, the composition of the photosynthetic apparatus refers only to Arabidopsis, which in this regard differs in some respects from Chlamydomonas. YCF3 and YCF4 (not shown) are localized on the thylakoid membrane, but not stably associated with a multiprotein complex. PC, plastocyanin; Fd, ferredoxin; FNR, ferredoxin-NADPH reductase; LHC, light-harvesting complex; PS, photosystem. [Leister Trends Genet 19:47] 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]

Plant mitochondrial genome coding content
In organello protein synthesis estimates 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)!

Table 3 General features of mtDNA of angiosperms
Plant mitochondrial genome coding content Table 3 General features of mtDNA of angiosperms Feature Ntaa Ath Bna Bvu Osa MC (bp) 430, , , , ,520 A+T content (%) Long repeated (bp) b 34,532 11,372 2,427 32, ,600 Uniquec 39, , , , ,065 Codingd (9.9%) (10.6%) (17.3%) (10.3%) (11.1%) Cis-splicing introns 25, , , , , (6.5%) (8.0%) (12.9%) (5.6%) (7.2%) ORFse 46, , , , , (11.8%) (10.4%) (9.2%) (16.1%) (3.3%) cp-derived (bp) 9,942 3,958 7,950 g 22, (2.5%) (1.1%) (3.6%) 2.1% h (6.2%) Others 274, , , , (69.3%) (69.9%) (57%) 65.9% (72.2%) Gene contentf aSee Table 2 for abbreviations of plant species names; bOne copy of each repeat is not considered ; cOne copy of each repeat is included. Lengths are expressed in bp and the percentage of unique sequence is shown in parentheses ; dStructural genes, rRNA genes and tRNA genes. The gene copy is not considered ; eLonger than 300 bp | fPseudogenes and gene copies are not considered | gData for cp-derived sequence from Handa (2003) ; hData for cp-derived sequence from Kubo et al. (2000) Gene content is similar but NOT identical. Why? (from Sugiyama et al. Mol Gen Gen 272:603)

Mitochondrial genomes encode integral membrane components of the respiratory complexes
II AOX intermembrane space inner membrane matrix I UQH2 UQ H+ CYC IV III ATP Synthase TCA cycle NADH NAD+ NAD(P)H DH external NAD(P)H DH internal 2H2O O2 ADP *** * **** * = one mitochondria-encoded subunit * (Modified from Rasmusson et al. Annu Rev Plant Biol 55:23)

Plastid genome transformation
What are the special challenges for the genetic transformation of organelle genomes?

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)

Figure 2. Biolistic chloroplast transformation and transgene integration into the plastid genome via homologous recombination. Left panel: The transformation vector is shot into living leaf cells on the surface of microscopic gold (or tungsten) particles. Small leaf pieces are subsequently exposed to a regeneration medium containing the plastid translational inhibitor spectinomycin. Because the transformation vector contains a spectinomycin resistance gene (aadA), transplastomic cells can regenerate despite the presence of spectinomycin. Primary transformants are usually heteroplasmic and, by passing tissue samples through several additional rounds of regeneration under antibiotic selection, residual wild-type genomes are eliminated. Finally, homoplasmic shoots are rooted in phytohormone-free medium. Right panel: Targeting of a transgene to a non-coding intergenic region of the plastid genome (ptDNA). The aadA gene is tethered to a plastid promoter and Shine–Dalgarno sequence (yellow) and a plastid 3′ untranslated region (UTR; red) conferring transcript stability. Transgene integration into the plastid genome occurs by two homologous recombination events in the flanking regions (dashed lines). Any transgene of interest can be co-incorporated by physically linking it to the aadA marker gene. The transgene of interest can either be driven by its own (plastid-derived) expression signals or combined with the aadA to form an operon. In the latter case, the transgene cassette consists of the following modules: Promoter→Shine–Dalgarno sequence 1→Coding region 1→Shine–Dalgarno sequence 2→Coding region 2→3′ UTR. [Bock & Khan, Trends Biotechnol 22:311]

Selection for plastid transformants
Figure 1. Generation of tobacco plants with transgenic chloroplasts A) leaf segments post bombardment with the aadA gene; B) leaf segments after selection on spectinomycin; C) transfer of transformants to spectinomycin + streptomycin to eliminate spontaneous spectinomycin resistant mutants; D) recovery of homoplasmic spec + strep resistant transformants after multiple rounds of regeneration on selective medium [Bock (2001) 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 [Bock , J Mol Biol 312:425]

Applications of plastid genome transformation
by homologous recombination Figure 1. Applications of chloroplast transformation technology and the corresponding design of plastid transformation vectors. (a) For reverse genetics, the selectable marker gene for plastid transformation aadA (a spectinomycin resistance gene) is used to disrupt an endogenous gene of unknown or unclear function. ycf, plastid open reading frame; ycfmut, mutated plastid open reading frame. (b) For gene replacement, an allele of an endogenous gene is linked to the aadA marker gene to replace the endogenous allele by homologous recombination. This strategy can also be used to introduce point mutations into plastid genes and open reading frames to study their functions. (c) For expression of a transgene of interest (GOI), this passenger gene is linked to the aadA gene and targeted to a neutral insertion site, typically an intergenic region of the plastid genome. A,B, flanking plastid DNA regions for integration via homologous recombination; ptDNA, plastid genome; UTR, untranslated region. [Bock , Curr Opin Biotechnol 18:100]

Functional analysis of plastid ycf6 in transgenic plastids
Knockout of the plastid ycf6 reading frame. (A) Properties of the putative Ycf6 protein. Alignment of the ycf6-derived amino acid sequences from higher plant species and the liverwort Marchantia polymorpha. Amino acid positions identical in all species listed here are denoted by asterisks. The hydropathy plot of the tobacco Ycf6 protein shows that the protein is highly hydrophobic and appears to consist of a single transmembrane domain (predicted to be largely of -helical conformation). DDBJ/EMBL/GenBank accession Nos are: Z00044 (N.t.), M55297 (S.o.), X86563 (Z.m.), X15901 (O.s.), D17510 (P.t.) and X04465 (M.p.). (B) Construction of a plastid transformation vector for targeted inactivation of ycf6. A map of the region of the tobacco plastid genome containing ycf6 and the map of the ptDNA targeting fragment in transformation vector pycf6 are shown. Genes above the line are transcribed from left to right, genes below the line are transcribed in the opposite direction. Relevant restriction sites are shown. Phenotype of ycf6 plants. A ycf6 plant is shown under standard light conditions (A) and under extreme low-light conditions (B). For comparison, a wild-type plant kept under identical low-light conditions is also shown (C). [Hager et al. EMBO J 18:5834]

Functional analysis of plastid ycf6 in transgenic plastids
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 Western blot analyses of thylakoid proteins from three independent transplastomic ycf6 lines and a dilution series of the wild type to test for the presence of key components of the protein complexes in the thylakoid membrane. Immunoblot analyses with antibodies against AtpB (CF1 subunit), PsaC, the plastocyanin-docking protein PsaF and the D1 and D2 proteins (PsbA and PsbD) confirm the presence of wild-type levels of chloroplast ATP synthase, PSI and PSII in ycf6 plants. Also, antibodies against the soluble electron carrier plastocyanin (PetE) detect similar levels of plastocyanin in extracted lumenal proteins from the wild type and the mutant. In contrast, immunoblots with anti-cytochrome f (PetA) antibodies revealed virtually a complete absence of cytochrome f protein from all ycf6 lines, suggesting that the mutants lack functional cytochrome b6f complex. Why, if ycf6 is the disrupted gene, does another PET complex subunit (PETA) fail to accumulate ? [Hager et al. EMBO J 18:5834]

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? A construct that consists of chloroplast sequences (C and D) that flank two selectable marker genes is inserted into the chloroplast genome through homologous recombination, thereby transforming the native plastome into a TRANSPLASTOME (a). In the experiments of Huang et al.83, the flanking chloroplast sequences were in the inverted repeats of the tobacco plastome. One of the selectable genes (aadA in the case illustrated) is designed for exclusive expression in the chloroplast and incorporation of this marker confers spectinomycin resistance. The other gene, a neomycin phosphotransferase gene neoSTLS2 (that encodes NPTII and incorporates a nuclear intron; here neo), is tailored, by virtue of a nuclear-specific promoter and the presence of a nuclear intron in the reading frame, for expression only when it is transposed to the nucleus. Continuous selection of growing leaf cells on spectinomycin medium allows transformed plastomes to be selected and eventually the transplastome entirely replaces the native chloroplast genome, such that all copies of the chloroplast genome contain the two selectable marker genes (b). Selection of cells or progeny seedlings on kanamycin medium allows the detection of the rare cases in which the neo gene has changed its location, such that strong expression is promoted from the nuclear environment (c). The progeny of self-fertilized transplastomic plants were not screened directly83. Rather, to eliminate low-level expression of neoSTLS2 from the chloroplast genome, transplastomic plants were crossed with wild-type female plants such that, because of strict maternal inheritance of tobacco plastids (Box 1), progeny that contained only wild-type chloroplasts were produced. Therefore, chloroplast-to-nucleus transposition must have occurred at some stage during the life cycle of the male parent of the seedlings that were screened on kanamycin plates. The observation that 1 in 16,000 male tobacco gametes contained a newly integrated segment of chloroplast DNA (Ref. 83) was unpredictably high, but it must be an underestimate of the true chloroplast-to-nucleus transposition frequency. In this experiment, the detection strategy enabled the identification only of those events that resulted in an entire, expressed neoSTLS2 gene in the nucleus. Other regions of the tobacco plastome that integrated in the nucleus without this selectable marker necessarily remained undetected. A similar strategy was used by Stegmann et al.84 and by Lister et al.85. [Timmis et al. Nat Rev Genet 5:123]

PCB6528 Plant Cell and Developmental Biology Spring 2015

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PCB6528 Plant Cell and Developmental Biology Spring 2015

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