Genome of plastids
and mitochondria
General features of P & M
• Double membrane
• Multiplication/reproduction by division
• Their own DNA and ribosomes (70S)
– Synthesis of only small portion of proteins
– Transfer of genes to nuclear genome
• Endosymbiotic origin
• Role in energetic metabolism
Origin of plastids and
mitochondria
*
The tree of Eucaryots has been updated!
Gillham 1994
Primary, secondary, tertiary endosymbiosis
Glaucophytes
– varying number of membranes (alt. nucleomorph)
Primary (1,6 BY) - Glaucophytes
- Rhodophytes
- Chlorophytes
Secondary
- Euglenophyta
- Chlorarachniophyta
- Chromalveolata
Transient endosymbiosis
(sea snail Elysia chlorotica)
- active chloroplasts of
sea weed Vaucheria
- active for up to eight months
- PsbO (MSP) present in Elysia
genome!!!
(Rumpho et al. 2008 PNAS)
Rumpho M. et al., 2000
Stromules of chloroplasts (tubular structures)
- contraversion if they allow exchange of genetic material and proteins
- clear communication with other organeles (ER, mitochondria?)
transfer NO
Schattat M et al. Plant Cell 2012;24:1465-1477
Hanson M, Sattarzadeh A. Plant Cell 2013;25:2774-2782: … transfer YES
Reproduction of plastids
Combination of procaryotic
FtsZ a eucaryotic
dynamine circle
Internal circle:
1. FtsZ
2. FtsZ + dynamin
3. dynamin
external: dynamin (role of ER?)
Lopez-Juez E., 2007
MITOCHONDRIA: FtsZ missing in plants, other factors, role of ER
Plastome a chondriome
versus nuclear genome
Gene functions in plastome (and chondriome)
+ genes for rRNA and tRNA (some are missing in mitochondria)
Nuclear encoded proteins in P&M
(predikce Target P u Arabidopsis – Emanuelsson et al., J Mol Biol)
Mitochondria ~ 10 % (cca 2500 genes)
Plastids
~ 14 % (cca 3500 genes)
Approx. 1/4 genes necessary for P&M
- many proteins do not originate from the endosymbiont, but host genome
or other endosymbiont
- many genes/proteins originating from endosymbionts used for various
functions in cytoplasm
Fate of genes
from the
endosymbiont
Arabidopsis
nucleome: approx. 26.000 genes
plastome: 87
proteins in plastid: ~ 3500 genes
endosymbiont (~ 4500 genes)
(blue-green algae:
~ 3000 – 7000 genes)
Leister D., TRENDS in Genetics 19: 47, 2003.
Causes of gene transfer to
nucleus
- more complex regulation of gene expression
- use of genes/proteins for secondary functions
- teoreticalla higher mutation speed in organells
- no recombination (correction of mutations) in sexual
reproduction
- however, chloroplast genes highly conserved!
Protein products have to be imported back to the
organelle!
Transport of proteins to plastids
Major pathway:
TOC-TIC translocons
(channels + chaperons!)
Transite peptide: 30-100
amk (2-4.000 proteins),
mitochondria: TOM-TIM
Inaba and Schnell; Biochem J (2008)
OM – outer membrane
ER-CP – glycoproteins
Uncleaved TP
Transport to thylakoids
Lumen: prokaryotic transport systems
Thylakoid membrane – spontaneous incorporation (+ plastid encoded
proteins)
Recent gene transfer
to nucleus
(if the gene obtains transite peptide, it
can be lost from the organelle)
• Fusion with transite peptide
of an imported protein
• e.g. mitochondrial Rps11
paralogs in rice use TP
of Cox and mitochondrial
ATPase subunit
(residues of mt gene are still transcribed)
Kadowaki et al., EMBO J. 1996
Why some genes are retained in
P&M?
- transmembrane proteins (complicated transport, folding –
cotranslational incorporation to membrane)
- high level and rapid protein synthesis
DNA transfer from organelles to
nucleus is still active!
- plant nuclear genomes contain high amount of plastid DNA
- even recent integrations (whole copies of plastid genomes)
- in rice totally more than 800 kbp
Transfer of genes from
organelles
high frequence of transgene transfer from
plastids to nucleus
- in somatic cells
1 of 18.000
- v pollen (plastid degradation!) 1 of 11.000
- in egg cells
1 of 250.000
Heritability of organelle DNA
- usually uniparental (maternal),
- various mechanisms:
–
Chlamydomonas: paternal organelle DNA eliminated through
methylation disabling its replication
(otherwise methylation of DNA and RNAi do not occur)
–
some higher plants – paternal plastids eliminated upon fertilization
Plastid DNA (cpDNA)
•
•
•
•
•
•
dsDNA, circular (likely basic from)
lower G-C content (compared to nucleus)
high copy number (~30-100) per plastid
20-40 organelles/nucleus
without histones, but Hu proteiny (Hu),
organized to nucleoids
10-20 % of total leaf DNA
Typical cp genome – basic arrangement
Circle divided into „long“
and „short“ unique regions
(LSC and SSC) separated
with IR (recombination =
inversion)
rRNA (rrn) and tRNA (trn)
genes in clusters (like in E.
coli)
Structural complexity of plastid DNA
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]
Sizes of plastid genome
• 70 - 200kb
• higher plants 120 – 170 kb
Sizes of mitochondrial genome
• S. cerevisiae
84 kb
• mammels
16 kb
• similar coding capacity
• higher plants – hunders kb
(x weed – small chondriome 16 kb)
Economization in evolution x
evolutionary trap in higher plants?
Mitochondrial genome
Mitochondrial
genome
Maize: Zea mays
several circle molecules
„master“ - 570 kb +
subgenomic molecules
derived from the „master“
Subgenomic circles
through recombination
between repeats (arrows)
Complexity of mt DNA
reason?
Backert et al. Trends Plant Sci 2:478
Expression of chloroplast genome
Genes mainly in operons – cotranscription
Polycistronic RNA
psbB
psbT
psbH
petB
Intron
psbN
Monocistronic RNA
petD
Intron
- processed to shorter segments
(present as stable ribonucleoprotein units)
Higher plants plastids – approx. 30 transcription units
(with promoter and terminater)
- two polymerases PEP, NEP (plastid, nucleus-encoded pol.)
- promoters for PEP similar to bacterial (–10 and –35 sequence)
- mostly promoters for both polymerases
transcript usually without cap and polyA, exceptionally
editing
Transcriptional units of
plastid genome
Model of expression of psbB operon
Barkan A. Plantphysiol 2011;155:1520-1532
- Important role of pentatricopeptide-repeat proteins (also involved in editing)
Expression of mitochondrial
genome
- transcripts without cap and polyA
- transkripts frequently edited
RNA Editing
– discovered in plant mitochondrial genes
– rare in plastids of higher plants
Definition: any process (except splicing) causing
change in RNA sequence (it is no more fully
complementary to the template DNA sequence)
Editing of mitochondrial transcripts
Exchange of C to U
cytosin deaminase or
replacement of
nucleotide base
•
•
•
Many mitochondrial transcripts (tRNA, protein-coding)
Mainly C to U
Guide RNA and editosom (role of PPR, mechanism?)
Introns in organelle genes
•
orthologous genes can have different introns in the
same positions
•
same or similar introns in various genes and
species (introns I. and II. type)
•
obtained and lost repeatedly during evolution
Expression of plastid genes
- PEP and NEP – (Plastid/Nuclear Encoded Polymerase)
- mutually regulated,
- widely overlaping expression (double promoters)
- not substitutible (both necessary)
- nuclear encoded sigma factors of PEP complex
- some  common for both plastids and mitochondria,
- transcription usually induced by more than one  factor
σ
Transcriptional regulation of
plastid gene expression
1. Global
– increase/decrease in expression of majority
of genes in the same time
2. Gene specific
sigma factors of PEP (procaryotic type)
e.g. psbD/psbC expression activated with light
Factors effecting chloroplast gene
expression
Retrograde signalling
- regulation of nuclear expression by signals from
P&M (products targeted to the organelles)
- response to changing conditions
- full expression only if fully functional plastids are present
Signals: - chlorophyll biosynthesis precursors
- electron transport components
- redox signals + ROS
- certain metabolites
Genes Encoded in the Chloroplast Genomes in Higher Plants
Gene Designation
Gene Product
I. Genetic System
Chloroplast RNA genes
rDNA
trn
Gene transcription
rpoA, B, C
ssb
Protein synthesis
rps2,3,4,7,8,11
rps12, 14, 15, 16, 18, 19
rpl2, 14, 16, 20, 22
infA
II. Photosynthesis
Photosynthetic proteins
rbcL
atpA, B, E
atpF, H, I
psaA, B, C
psbA, B, C, D, E
psbF, G, H, I
petA, B, D
Respiratory proteins
ndhA, B, C, D
ndhE, F
III. Others
Maturase
Protease
Envelope membrane protein
Ribosomal RNAs (16S, 23S, 4.5S, 5S)
Transfer RNAs (30 species)
RNA polymerase a, b, b’ subunits
ssDNA-binding protein
30S ribosomal proteins (CS) 2, 3, 4, 7, 8, 11
CS12, 14, 16, 18, 19
50S ribosomal proteins (CL) 2, 14, 16, 20, 22
Initiation factor I
RUBISCO large subunit
ATP synthetase CF1a, b, e subunits
ATP synthetase CF0I, III, IV subunits
Photosystem I A1, A2, 9-kDa protein
Photosystem II D1, 51 kDa, 44 kDa, D2, Cytb559-9kDa
Photosystem II Cytb559-4kDa, G, 10Pi, I proteins
Electron transport Cytf, Cytb6, IV subunits
NADH dehydrogenase (ND) subunits 1, 2, 3, 4
NDL4L, 5
matK
clpP
cemA