Transcript PowerPoint 1

PCB6528 Plant Cell and Developmental
Biology Spring 2012
Organelle genomes, gene expression and
signaling
Christine Chase – 2215 Fifield Hall –
352-273-4862
[email protected]
Organelle genomes
Organelle gene expression processes
Organelle-to-nucleus signaling
(retrograde regulation)
Objectives - Organelle genomes:
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 plastid or mitochondrial genome coding
content is not necessarily 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
Small but essential genomes
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 maternally inherited in plants
Encode necessary but insufficient information to
elaborate a fully functional organelle
Many nuclear gene products required for
organelle function
translated on cytosolic ribosomes &
imported into the organelles
plant mitochondria also import tRNAs
needed for a complete set!
Considerable cross-talk between nuclear and
organelle genetic systems
Comparative sizes of plant genomes
Genome
Size in bp
Arabidopsis thaliana
1.4 x 10
8
3.7 x 10
5
nuclear
Arabidopsis thaliana
mitochondria
Arabidopsis thaliana
plastid
Zea mays
nuclear
Zea mays
mitochondria
Zea mays
plastid
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 and 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 the eukaryotic genomes
Reduced coding content of organelle genomes
•Functional gene transfer to nucleus with
protein targeted back to organelle
•Functional re-shuffling - organelles replace
prokaryotic features with eukaryotic, “hybrid”
or novel features
Evolution of mitochondrial genome coding content
Genome
Protein
coding
genes
Rikettsia prowazekii
832
(smallest  proteobacterial
genome)
Reclinomonas americana
62
Marchantia polymorpha
64
Arabidopsis thaliana mitochondria
57
mitochondria
(protozoan; most mitochondrial
genes)
mitochondria
1.9 x 10 5 bp
(liverwort, non-vascular plant )
3.7 x 10 5 bp
(vascular plant)
Homo sapiens mitochondria
13
Evolution of plastid genome coding content
Genome
Protein
coding genes
Synechococcus (cyanobacteria)
3,300
Paulinella chromatophora
867
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)
209
63
67
71
42
Functional gene transfer from organelle
to nuclear genome
• Gene by gene
• Likely occurs via RNA intermediates
• 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 relocation from organelle to nucleus?
• How would we know this occurs via RNA
intermediates?
Functional gene transfer: Recent repeated
transfers of the plant mitochondrial rps10
to the nucleus
•Southern blot hybridization of total cellular DNA
samples
• Mitochondrial nad1 and rps10 probes
• Shading = taxa with no hybridization to rps10
• Bullets = taxa with confirmed nuclear copy of rps10
[Adams et al. Nature 408:354]
• Why is there no hybridization of rps10 probes to DNA
samples with confirmed nuclear copy of rps10?
(Hint: How are the relative genome copy numbers and
sizes exploited in this screen?)
•What is the purpose of the nad1 probe?
•What are the consequences of these events with respect
to plant mitochondrial genome coding content?
Reduced plastid genome content in
nonphotosynthetic plastids:
Parasitic plants with 70-20 kb plastomes have lost
photosynthetic genes, some ribosomal proteins &
some tRNAs
Essential tRNA hypothesis: [Barbrook et al. Trends
in Plant Sci. 11:101]
Plastid tRNAs needed to support mitochondrial
function
• tRNA-Glu as precursor for the synthesis of heme
for mitochondrial respiratory electron transfer
• tRNA-Met imported into mitochondria for
mitochondrial protein synthesis
Epifagus virginiana
(beechdrops)
A non-phoptosynthetic,
parasitic plant
Has a plastid genome of 71
kb encoding 7 tRNAs and 2
ribosomal proteins
http://2bnthewild.com/plan
ts/H376.htm
Land Plant Plastid Genome Organization
Physical map (e. g. restriction map or DNA
sequence) indicates a 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
Inversion polymorphisms within single copy
regions mediated by small dispersed repeats
Plastid genome organization
(Maier et al. J Mol Biol
251:614)
Plastid 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)
Recombination across inverted repeats
leads to inversions
rps15
ndhF
trn N
trn N
ndhB
ndhB
rps19
rps19
rpl22
psbA
How can
these
inversion
isomers be
detected?
ndhF
trn N
ndhB
rps19
psbA
rps15
trn N
ndhB
rps19
rpl22
Fiber FISH of tobacco plastid DNA
[Lilly et al. Plant Cell. 13:245]
Structural Plasticity of cpDNA Molecules
from Tobacco, Arabidopsis, and Pea
[Lilly et al. Plant Cell. 13:245]
Structural Plasticity of cpDNA Molecules
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]
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]
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 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 (nuclearlocalized mitochondrial DNA)
88,000 years ago
e.g. Rice 131 kb nupDNA (nuclear-localized
plastid DNA)
148,000 years ago
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]
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
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 detected?
Arabidopsis mitochondrial genome organization
>
>
>
>
>
Two pairs of repeats active in recombination
•One direct (magenta, top left)
•One inverted (blue, top left)
Recombining the inverted (blue pair) creates an
inversion
•What has happened to the orientation of the
magenta 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 plasticity of plant mitochondrial
DNA
[Backert et al. Trends Plant Sci 2:478]
Structural plasticity 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 – 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]
Recombination initiated DNA replication
[Kreuzer et al. J Bacteriol 177:6844]
Possible origins of structural plasticity in
plant organelle genomes
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
• recombination-mediated replication
Few if any genome-length circular molecules
• limited number of circular rolling circle
replication templates
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
(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
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)