Transcript Organelle genomes

PCB6528 Plant Cell and Developmental
Biology Spring 2016
Organelle genomes, gene expression and
signaling
http://www.hos.ufl.edu/sites/default/files/courses/
pcb6528/PCB6528S16_outline.htm
Christine Chase – 2215 Fifield Hall –
352-273-4862
[email protected]
How nuclear and cytoplasmic genetic
systems co-evolved and now interact to
build and maintain highly abundant and
complex respiratory and photosynthetic
factories
[Leister, Trends Genet 19:47; Salvato, Plant Physiol 164:637]
Objectives - Organelle genomes:
Describe the organization and coding
content of modern-day organelle genomes
Reconcile discrepancies between physical
maps and the observed DNA structures
Describe how evolution has shaped and
changed modern-day organelle genome
coding content compared to ancestral
prokaryotic genomes
Consider the possible reasons that plant
organelles retain genomes at all!
Explain the challenges and experimental
opportunities associated with genetic
transformation of the plastid genome
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 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
Genome
Size 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
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]
Eukaryotic nuclear genomes – a big mix &
match experiment
383 eubacterial- &
111 archeaebacterialrelated genes in the
yeast nuclear genome
Genes per category
Esser et al. 2004 Mol
Biol & Evol 21:1643
Evolution of mitochondrial genome coding content
Genome
Protein
coding
genes
Rikettsia prowazekii
(smallest  proteobacterial
genome)
832
Reclinomonas americana
mitochondria
(protozoan)
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
Saccharomyces cerevisiae
7.8 x 10 4 bp
(yeast)
11
Homo sapiens mitochondria
13
Evolution of plastid genome coding content
Genome
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 modern-day 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]
•
•
•
•
•
The structures of nuclear genes encoding
mitochondrial RPS10
• Triangles = nuclear intron positions
• Shading = mitochondrial targeting sequences
• Stripes = regions homologous to other known
nuclear genes.
• Arrow = continuing open reading frame
• What do these diverse gene structures, along
with phylogeny, tell us about the functional
transfer of the rps10 gene from mitochondria to
nucleus?
29 of the 31 mitochondria-located rps10 genes
examined in this study contain a conserved intron,
which is missing in all of the nuclear-located rps10
genes. What does this suggest about the mechanism
of functional gene transfer from mitochondria to
nucleus?
[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
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
rps15
ndhF
trn N
trn N
ndhB
ndhB
rps19
rps19
rpl22 How can
psbA
these
inversion
isomers be
distinguished?
ndhF
trn N
ndhB
rps19
psbA
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
Structurea
Arabidopsis
Tobacco
Pea
Circular
126 (42%)
524 (45%)
59 (25%)
Linear
68 (23%)
250 (22%)
85 (36%)
Bubble/D-loop
25 (8%)
67 (6%)
5 (2%)
Lassolike
34 (11%)
115 (10%)
21 (9%)
Unclassifiedb
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
• Almost no conservation of gene order among
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 maps (and
structures)
Recombination across direct repeats
leads to deletions (subgenomic molecules)
a
c
b
d
PmeI
Not I
AscI
c’ b’
d’ c’
b
a
c
Pac I
d
b’ c’ d’
Pac I
Not I
a
AscI
b’ a’
b
c’
d’
AscI
Pac I
c
d
PmeI
How can these deletion (subgenomic) isomers be
distinguished?
Arabidopsis mitochondrial genome organization
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) While 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
(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
for mitochondria
Circular maps from linear molecules
A
Z
Y
B
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]
Circular maps from linear molecules
A
Z
Y
B
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
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
wild-type recombination partners
far out-number a new mutant !
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
• Prevents or limits recombination
between short repeats (~100-500 bp) in
plant organelle DNAs
Mediated by four nuclear-encoded
protein families
• Dual-targeted or duplicate genes
encode pt- & mt-targeted versions
• MSH1 - Eubacterial mismatch repair
homologs
• RECA - Eubacterial recombination
homology search & strand invasion
• OSB - Recently evolved plant-specific
organelle single-stranded DNA binding
proteins
• Whirly - Recently evolved, primarily
plant, single-stranded DNA binding
proteins
Plant organelle recombination
surveillance
DSB in mtDNA
RECA, ODB/RAD52
WHY
homologous
recombination/
gene conversion
RECA, MSH1,
OSB
RECA-independent
recombination
at micro-homologies
Mutations in any surveillance gene destabilize mitochondrial and/or plastid
genome organization
[Modified from Gualberto et al. Biochimie 110:107]
Down-regulation of dual-targeted MSH1
alters organelle function and genome
organization
Mitochondrial genome reorganization
left, co-segregating with leaf
variegation, right
• Organelle recombination is regulated
• Regulated homologous recombination
important for genome replication and
repair
• De-regulation destabilizes organelle
genome organization with phenotypic
consequences
[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
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 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
Ntaa
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
Uniquec
Codingd
37,549
(10.6%)
38,065
(17.3%)
34,499
(10.3%)
40,065
(11.1%)
Cis-splicing introns 25,617
(6.5%)
28,312
(8.0%)
28,332
(12.9%)
18,727
(5.6%)
26,238
(7.2%)
ORFse
46,773
(11.8%)
37,071
(10.4%)
20,085
(9.2%)
54,288
(16.1%)
12,009
(3.3%)
cp-derived (bp)
9,942
(2.5%)
3,958
(1.1%)
7,950 g
(3.6%)
2.1% h
22,593
(6.2%)
Others
274,527 248,662 124,994
(69.3%) (69.9%) (57%)
65.9%
262,015
(72.2%)
Gene contentf
60
52
56
39,206
(9.9%)
55
53
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
NAD(P)H DH
external
H
+
UQH2
****
***
inner
membrane
CYC H
+
H
+
III
I
UQ
*
*
NAD+
NADH
ATP
Synthase
* ***
2H2O
IIII
TCA
cycle
****
IV
O2
AOX
NAD(P)H DH
internal
intermembrane
space
2H2O
O2
H
+
ADP
*
matrix
ATP
= 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?
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]
Applications of plastid genome transformation
by homologous recombination
[Bock , Curr Opin Biotechnol 18:100]
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]