Small but essential genomes

1
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 proteins 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
2
Comparative sizes of plant genomes
3
Organelle genomics proteomics
Target P prediction analysis of the complete
Arabidopsis nuclear genome sequence says
..... About 10 of the Arabidopsis nuclear
genome (2,500 genes) encode proteins targeted to
the mitochondria About 14 of the Arabidopsis
nuclear genome (3,500 genes) encodes proteins
targeted to the plastid Hence 25 of the
Arabidopsis nuclear genome is dedicated to
organelle function Proteomic study of
Arabidopsis mitochondria estimates 1,500 2,000
proteins Proteome reflects metabolic diversity
of these organelles, both anabolic and catabolic
4
Diverse plastid forms and functions

Primary site of anabolic reactions Photosynthesis
Amino acid biosynthesis Starch biosynthesis and
storage amyloplasts Carotenoid biosynthesis and
storage chromoplast Lipid biosynthesis and
storage leucoplast
Images from Buchanan et al. 2000 Biochemistry and
Molecular Biology of Plants
5
Diverse mitochondrial functions
Catabolic metabolism TCA cycle Respiration Oxidat
ion of sugars and fatty acids Anabolic
reactions Heme, Fe-S complexes, Ubiquinone,
Folate,
Image from Burger et al. Trends in Genet 19
709-716
6


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 211643
8
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
• Non-functional transfer of DNA
• mit to nucleus
• plastid to nucleus
• plastid and nucleus to plant mit

Figure 1   Organellar DNA mobility and the
genetic control of biogenesis of mitochondria and
chloroplasts.  From Timmis et al. (2004) Nature
Rev Genet 5123
9
Evolution of mitochondrial genome coding content

10
Evolution of plastid genome coding content

11
Functional gene transfer from organelle to
nuclear genome

• Gene by gene
• Likely occurs via spliced and edited 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
  re-location from organelle to nucleus?

12
An example of functional gene transfer Recent
repeated transfers of the plant mitochondrial
rps10 to the nucleus
Southern blot hybridization of total cellular DNA
samples with mitochondrial nad1 and rps10 exon
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
408354 Explain why there is no hybridization
of rps10 probes to DNA samples found to contain a
nuclear copy of rps10? (i.e. How are the relative
genome copy numbers and sizes exploited in this
screen?) What is the purpose of the nad1 probe?
13
Evolutionary trend towards reducing organelle
gene content

Fig. 4 Loss of genes from the mtDNA during
evolution from Marchantia to antiosperms.
Phylogenetic relationships for antiosperms are
based on published results (Soltis et al. 1999).
The order of gene loss in each lineage is not
clear. From Sugiyama et al. (2005) Mol Gen
Genomics 272603
14
An example of functional re-shuffling of
eukaryotic, prokaryotic and organelle features
plastid division

• Inner division ring established by
  prokaryotic-type FTSZ (tubulin) guided by MIN and
  DNAJ (ARC6) proteins
• Outer division ring and final constriction
  accomplished by ARC5, similar to the dynamin
  eukaryotic membrane severing protein

from Lopez-Juez and Pyke (2005) Int J Dev Biol
49 557
15
Non-Functional DNA transfer from organelle to
nuclear genome

• Frequent
• Continuous (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

16
Why have organelles retained genomes?

17
Reduced plastid gene 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. 11101
• 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/plants
/H376.htm
18
The hydrogenosome mitochondria without a
genome?

Membrane-bound organelle found in some anaerobic
protozoans Synthesizes ATP anaerobically
Contains nuclear-encoded mitochondrial
chaperones HSP10, HSP60 and HSP70
Nycototherus ovalis hydrogenosome contains a
rudimentary genome encoding mitochondrial-type
complex I subunits ? Genome loss with loss in
the absence of respiratory function
from Gray (2005) Nature 43429
19
Why have functional organelles retained genomes?

20
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
• Conifers and some legumes have no LIR
• Inversion polymorphisms within single copy
  regions mediated by small dispersed repeats

21
Plastid genome organization (from Maier et al.
JMB 251614)
22
Recombination across inverted repeats leads to
inversions
23
Structural Plasticity of cpDNA Molecules from
Tobacco, Arabidopsis, and Pea
(B) tobacco chloroplast fibers hybridized with an
IR probe (red FISH signal) (C) monomeric and
multimeric cpDNA molecules from tobacco (red
signals - IR probe) (D) linear hexameric tobacco
molecule (green signals represent LSC red
signals - SSC IR) (E) circular tetrameric
chloroplast molecule from Arabidopsis (F) Two
monomeric circular molecules from pea (red signal
origin of replication probe) Bars 10 µm
Lilly et al. Plant Cell. 13245-54
24
Structural Plasticity of cpDNA Molecules from
Tobacco, Arabidopsis, and Pea
Lilly et al. Plant Cell. 13245-54
25
Plastid genome coding content
Generally conserved among land plants, more
variable among algae Genes involved in 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) Organized in
operons some gene orders conserved with bacteria
26
Plastid genomes encode integral membrane
components of the photosynthetic complexes
Photosynthetic composition of the thylakoid
membrane Green plastid-encoded subunits Red
nuclear-encoded subunits Leister, D. Trends
Genet 1947
27
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)
28
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
mutant 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)
29
Plastid genome transformation
Bock Khan (2004) Trends Biotechnol 22311
30
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 312425
31
Advantages of plastid genome transformation
High levels of expression Plastid proteins the
most abundant in the world No apparent gene
silencing in plastids Codon preference Bacterial
codon preferences used in the plastid Useful
bacterial genes can be expressed efficiently
without re-engineering codon usage antibiotic
resistance, herbicide resistance, insect
resistance, etc. Containment plastid genes are
not expressed in the pollen (although plastid
genomes may be present in some species)
eliminates pollen toxicity Plastid genomes are
maternally transmitted in many (but not all)
plant species Decreased pollen transmission of
transgenes to neighboring wild or cultivated
plants Important research tool Precise gene
targeting by homologous recombination
32
Applications of plastid genome transformation by
homologous recombination
Bock (2007) Curr Opin Biotechnol 18100
33
Functional analysis of plastid ycf6 in
transgenic plastids
Hager et al. (1999) EMBO J 185834
34
Functional analysis of plastid ycf6 in
transgenic plastids

• ycf6 knock-out lines
• Homoplasmic for aadA insertion into ycf6 via PCR
• Pale-yellow phenotype
• Normal PSI function and subunit accumulation
• Normal PSII function and subunit accumulation
• Abnormal b6f (PET) complex subunit accumulation
• Purification and mass spectrometry analysis of
  normal plastid PET complex demonstrates the
  presence of YCF6

Why, if ycf6 is the disrupted gene, does another
PET complex subunit (PETA) fail to accumulate ?
Hager et al. (1999) EMBO J 185834
35
Non-functional plastid-to-nucleus DNA transfer
Box 4 Design of experiments that showed DNA
transfer from organelles to nucleus in real time
a transform plastids with spec resistance
gene (aadA gene with a plastid promoter) linked
to Kan resistance (neo gene with a nuclear
promoter) b recover spec resistant plants
and use them to pollinate wild-type plants
c germinate seed from b on kanamycin to recover
kan gene transferred from plastid to nucleus
prior to pollination in b 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. (2004) Nat Rev
Genet 5123

36
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

37
Recombination across direct repeats leads to
deletions
PmeI
Not I
AscI
Pac I
AscI
Not I
PmeI
Pac I
How can these deletion (subgenomic) isomers be
detected?
38
Recombination across direct repeats leads to
deletions
39
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
2478
40
Physical structures of plant mitochondrial DNA
modified from Backert et al. Trends Plant Sci
2478
41
Reconcile physical (restriction) maps with
observed DNA structures

• Plastid genomes map as a single circle
• 2 inversion isomers imply recombination through
  the large inverted repeat
• Plant mitochondrial genomes map as a single
  master circle
• Many subgenomic circles and inversion isomers
  imply recombination through multiple direct and
  inverted repeat pairs
• Direct visualization of DNA from each of these
  organelles via EM or fluorescence in situ
  hybridization reveals
• Complex rosette/knotted/branched structures
• Longer-than genome linear molecules
• Branched linear molecules
• Few if any genome-length circular molecules
• Shorter-than genome linear and circular
  molecules
• Sigma molecules

42
Circular maps 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. These linkages also hold true
for the following 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
43
Physical structures of DNA obtained via rolling
circle DNA replication (from Freifelder, 1983,
Molecular Biology)
44
Recombination initiated DNA replication
From Kreuzer et al. J Bacteriol 1776844
45
Reconcile physical (restriction) maps with
observed DNA structures
Complex rosette/knotted structures ?
nucleoids Longer-than genome linear molecules ?
rolling circle replication ? intermolecular
recombination between linear molecules Branched
linear molecules ? recombination ?
recombination-mediated replication Few if any
genome-length circular molecules ? limited
number of circular rolling circle replication
templates Shorter-than genome linear and
circular molecules ? intramolecular recombination
between direct repeats Sigma molecules ? rolling
circles ? recombination between circular linear
molecules
46
Plant mitochondrial genome coding content
In organello protein synthesis estimates 30-50
proteins encoded by plant mitochondrial
genomes Complete sequence of A. thaliana
mitochondrial genome identified 57 genes
respiratory complex components rRNAs, tRNAs,
ribosomal proteins cytochrome c biogenesis
Plant mitochondrial genomes do not encode a
complete set of tRNAs mit encoded tRNAs of
native (mitochondrial origin) mit encoded tRNAs
derived by functional transfer from the plastid
genome missing tRNAs are nuclear encoded and
imported into mitochondria to complete the
set 42 orfs in A. thaliana mit genome that might
be genes A. thaliana mitochondrial gene density
(1 gene per 8 kb) is lower than the nuclear gene
density (1 gene per 4-5 kb)!
47
Plant mitochondrial genome coding content
Table 3 General features of mtDNA of angiosperms
(from Sugiyama et al. (20054) Mol Gen Gen
272603) Feature Ntaa Ath Bna Bvu Osa MC
(bp) 430,597 366,924 221,853 368,799 490,520 AT
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 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) ORFse 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) Others 274,527 248,662
124,994 262,015 (69.3) (69.9) (57)
65.9 (72.2) Gene contentf 60 55 53 52 56 aSee
Table 2 for abbreviations of plant species names
bOne copy of each repeat is not considered
cOne copy of each repeat is included. Lenghts 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)
48
Mitochondrial genomes encode integral membrane
components of the respiratory complexes

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?
49
Cytoplasmic male sterility (CMS) Maternally
inherited failure to produce or shed functional
pollen Observed in gt 100 different plant
species Conditioned by a variety of mosaic
genes that arise by recombination events in the
plant mitochondrial genome Novel mitochondrial
signaling pathways that condition degeneration or
homeotic transformation of male floral organs
Suppressed by nuclear restorer-of-fertility
alleles
50
Mosaic nature of mitochondrial CMS genes
Hanson Bentolila (2004) Plant Cell 16S154
51

• Mechanisms of CMS
• Homeotic phenotypes
• petaloid (stamens into petals)
• carpeloid (stamens into carpels)
• loss of B class MADS box expression
• retrograde regulation of nuclear gene expression
  via organelle signals
• Degenerative phenotypes
• apoptotic like death of tapetal cells and/or
  developing pollen
• necrotic like death of tapetal cells and/or
  developing pollen

52
Homeotic mechanisms of CMS
53

• Significance of CMS
• A non-lethal mitochondrial mutation
• Functions as a mitochondrial reporter gene
• Note there is currently no system for genetic
  transformation of plant mitochondrial genomes
• CMS /- fertility restoration genes provide a
  means to dissect nuclear regulation of
  mitochondrial gene expression
• An opportunity to investigate plant
  mitochondrial signaling pathways
• Precise control of pollen fertility for
• Commercial F1 hybrid seed production
• Reduction or elimination of nuclear transgene
  escape via pollen

54

• Mechanisms of fertility restoration
• Rf2 of CMS-T maize
• encodes a functional mitochondrial aldehyde
  dehydrogenase
• restoration via metabolic compensation
• All others - failure to accumulate CMS gene
  product
• restoration via changes in mitochondrial gene
  expression
• transcriptional or post-transcriptional
• To date, all of these encode pentatricopeptide
  repeat proteins