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
Organelle genomes
Organelle gene expression processes
Organelle-to-nucleus signaling
(retrograde regulation)

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

Organelle genomes
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

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

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]

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

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

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

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

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
• 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
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

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

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)

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

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

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

Structural complexity of plastid DNA from tobacco, arabidopsis, and pea
Table 1. Frequency of Different cpDNA Structures across All Experiments in Three
Species
No. of Observations
Structure a Arabidopsis Tobacco Pea
Circular
Linear
Bubble/D-loop
Lassolike
126 (42%)
68 (23%)
25 (8%)
34 (11%)
524 (45%)
250 (22%)
67 (6%)
115 (10%)
59 (25%)
85 (36%)
5 (2%)
21 (9%)
Unclassified b 44 (16%) 203 (17%) 66 (28%) a Each classification represents all molecules of that type regardless of size. b DNA fibers that were coiled or folded and could not be classified
[Lilly et al. Plant Cell. 13:245]

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

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

Arabidopsis mitochondrial genome organization
> >
>
>
>
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)?
[modified from Backert et al. Trends Plant Sci 2:478]

Branched rosette and linear molecules from
C. album mitochondria
(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
• 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)

Circular maps from linear molecules
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 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]

Recombination dependent DNA replication
[RDR]
[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
• 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

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

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

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)

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 nuclearencoded 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 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)!

Plant mitochondrial genome coding content
Table 3 General features of mtDNA of angiosperms
Feature Nta a Ath Bna Bvu Osa
MC (bp) 430,597 366,924 221,853 368,799 490,520
A+T content (%) 55.0
55.2
54.8
56.1
56.2
Long repeated (bp) b 34,532 11,372 2,427 32,489 127,600
Unique c
Coding d
39,206 37,549 38,065 34,499 40,065
(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 cp-derived (bp)
Others
Gene content f
46,773 37,071 20,085 54,288 12,009
(11.8%) (10.4%) (9.2%) (16.1%) (3.3%)
9,942 3,958
(2.5%) (1.1%)
7,950 g 22,593
(3.6%) 2.1% h (6.2%)
274,527 248,662 124,994 262,015
(69.3%) (69.9%) (57%) 65.9% (72.2%)
60 55 53 52 56
(from Sugiyama et al. Mol Gen Gen 272:603)

Mitochondrial genomes encode integral membrane components of the respiratory complexes
NAD(P)H DH external
H
+ inner ***
I
NAD(P)H DH internal
H
+
CYC
H
+ intermembrane space
UQH
2
UQ
*
TCA
NAD+ cycle NADH
AOX
*
III IV
***
O
2
2H
2
O
H
+
2H
2
O
O
2
Synthase
ADP ATP
* matrix
* = one mitochondria-encoded subunit
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)

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)

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

Selection for plastid transformants
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
[Bock , Curr Opin Biotechnol 18:100]

Functional analysis of plastid ycf6 in transgenic plastids
[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
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?
[Timmis et al.
Nat Rev Genet 5:123]