Summary - plant organelle genomes
Feature
plastid
plant mitochondria
size
conserved ~120160 kbp
crazy - 200-2,400
kbp
physical maps
circle
master circle
2 inversion
isomers
inversion isomers
recombination
repeats
conserved LIR
not conserved
inverted and direct
recombination
surveillance
shared
subgenomic circles
prokaryotic: MSH, RECA
plant specific: WHIRLY, OSB
observed
structures
circular monomer variable circular
linear and
> 1 genome linear
circular multimer
branched linear
branched linear
lariat
lariat
coding
conserved
conserved
gene order and
igs
conserved
crazy - not at all
conserved
genetic
transformation
yes
no
Objectives - Organelle gene expression
& signaling:
Describe the molecular processes that lead from
the organelle genes to fully assembled multi-subunit
protein complexes and understand the molecular
approaches that can be used to investigate these
processes
Describe how prokaryotic and newly evolved
eukaryotic proteins cooperate in organelle gene
expression processes
Define retrograde regulation and discuss how
organelle signals can adapt gene expression t
optimize organelle functions to changing
environments
What are the processes needed to take
us from gene to fully functional, multisubunit, organelle protein complex?
Consider both organelles at the same time
• Many shared processes and players
?Can organelle DNA copy number can
influence gene expression levels
1- No correlation of mitochondrial gene
copy number and transcript
accumulation in Arabidopsis leaves
(Preuten et al. The Plant J 64:948)
2 – Mitochondrial orf239 in Phaseolus
vulgaris might be a special case
• cytoplasmic male sterility (CMS) gene
locates on a subgenomic molecule
• high copy number > CMS
• reduced copy number > pollen fertility
• copy number mediated by nuclear
gene – Fr
(Mackenzie and Chase Plant Cell 2:905)
RNA Polymerases and promoters
Polymerase
Subunits
Consensus promoter
αββ’ β’’& σ 70
-35/-10
GTGTTGACA/TATAA
TG
Plastid –
encoded
PEP
αββ’ &
nuclearencoded σ
specificity
-35/-10
-TTGACA/TATAAT
Phage T7
single core
no σ
overlaps initiation
ATACGACTCACTATA
GGGAGA
Bacterial
Nuclear encoded
T7-like core &
plastid
+/- specificity
NEP/RPOTp
factor
Nuclear –
encoded
mit RPOTm
T7-like core &
+/- specificity
factor
overlaps initiation
ATAGAAT A/G AA
overlaps initiation
CRTA G/T
Differential plastid gene expression based
upon recognition of distinct promoters
by NEP and PEP
Most plastid genes have promoters for both
polymerases
Genes encoding expression machinery (e.g.
rpo, rrn, rps, rpl) primarily transcribed by
NEP
Photosynthetic genes primarily transcribed
by PEP
(from Hajdukiewicz et al. EMBO J 16:4041)
RNA Polymerases and promoters
What strategy could you use to identify
consensus promoters?
Gene 1
DNA
RNA 5’ ppp
5’ RACE or
5’
circular RT-PCR
locate initiated 5’ end
3’
3’
Gene 2
DNA
RNA 5’ ppp
5’ RACE or
5’
circular RT-PCR
locate initiated 5’ end
3’
3’
Gene 3
DNA
RNA 5’ ppp
5’ RACE or
5’
circular RT-PCR
locate initiated 5’ end
3’
3’
Organelle transcripts - initiated vs.
processed 5’ ends
• Organelle transcripts are not capped in vivo
• Initiated transcripts have 5’ tri-phosphate
PP- can be removed by tobacco acid
phosphatase (TAP) allowing ligation to an
adaptor for PCR & sequence
* processed 5’ end
*
Organelle transcripts - initiated vs.
processed 5’ ends
Processed transcripts 5’ mono-phosphate
Substrate for ligation
e.g. RNA adapter for 5’ RACE + PCR
Initiated transcripts 5’ tri-phosphate
Ligate only after de-phosphorylation
tobacco acid pyrophosphatese (TAP)
Compare PCR products +/—TAP
initiated transcript –not a ligation substrate
5’PPP
RNA
3’
processed or TAP-treated transcript
RNA
adaptor
Adaptor
primer
RNA
P
cDNA
PCR product
3’
3’ Gene
3’ primer
PCR products containing initiated 5’ ends
appear only after TAP treatment
Identification of promoters in
Arabidopsis plastids
+ T: with tobacco acid pyrophosphatase treatment
- T: no pyrophosphatase treatment
g: green tissue
w: white tissue (seedlings grown on spectinomycin)
[Swiatecka-Hagenbruch Mol Genet Genomics 277:725]
Diversity of promoters in
Arabidopsis plastids
[Swiatecka-Hagenbruch Mol Genet Genomics 277:725]
Plasticity of promoters in
Arabidopsis mitochondria
+TAP
- TAP
[Kühn et al. Nucleic Acids Res. 33:337]
Plasticity of promoters in
Arabidopsis mitochondria
Consensus for 20 sequences supporting
initiation at A
Consensus of 11 sequences supporting
initiation at G
[Kühn et al. Nucleic Acids Res. 33:337]
Multiple sigma factors of A.
thaliana with different plastid
promoter targets
Transcripts ↑
Transcripts ↓ in Sig knock downs w/ Sig over
expression
−Sig2 −Sig4 −Sig5 −Sig6 +Sig2 +Sig5
LRPatpBEtrnEYD ndhF
trnEYD psaA
psbDb 2.6kbb
trnV
psbAc psbA
psbA
trnM
psbBc
psbB
psaJ
psbCc
psbD
psbAa
psbDc
constpsbHc
b
psbD
psbNc
psbTc
rbcLc
rrn16c
rrn23c
rrn5c
rrn4.5c
• SIG2 and SIG6 are essential – knock outs are
chlorophyll deficient
• SIG 5 - expression induced specifically by blue-light &
recognizes a plastid blue light responsive promoter
[Lysenko, Plant Cell Rep. 26:845]
Redox regulation of plastid
transcription is adaptive
PSI
PSII
FD
PET
Light II
PSII most efficient
PSI less efficient
Additional PSI (psa)
subunits needed
PQ highly reduced
(as in + DBMIB)
FD highly oxidized
Light I
PSI most efficient
PSII less efficient
Additional PSII (psb)
subunits needed
PQ highly oxidized
(as in + DCMU)
FD highly reduced
[Surpin, Plant Cell Supplement 2002:S327]
Regulation of plastid transcription
through plastid redox signals
PSI
PSII
Why do the curves for relative transcript amounts
and relative transcription activity differ? What do
these two things measure?
Complementary changes in transcription rate and
mRNA abundance for psaAB (photosystem I) and
psbA (photosystem II) during acclimation to light I
or light II can be mimicked with DCMU and DBMIB
[Pfannschmidt et al. Nature 397:625]
Regulation of plastid transcription
through plastid redox signals
- SIG1 phosphorylation
In SIG1 T170 is a
phosphorylation site
SIG1 T170V mutant no
phosphorylation site
DBMIB > PQred
low psb/psa optimium
SIG1 nonphosphorylated &
very efficient for
psaA promoter
DCMU > PQox
high psb/psa
optimum
SIG1 phosphorylated
& less efficient for
psaA promoter
Shimizu Proc Natl Acad Sci USA 107:10760
Plant organelle RNA metabolism
Plant organelle genes are often cotranscribed
• Plastid operons
• Mitochondria – di-cistronic transcripts
In contrast to prokaryotic transcripts,
plant organelle transcripts require
modification prior to translation:
• Many are processed to di or monocistronic transcripts
• Many contain introns
• Many must undergo RNA editing to
encode functional proteins
Plant organelle RNA metabolism:
psbB operon processing in maize
How did we come to know all of this?
Step 1 - Forward genetics
• mutant screens
• phenotyping for plastid transcripts & proteins
Step 2 - Search for interacting genes & factors
• antibody development and co-immunoprecipitation
• yeast two hybrid screens
• tight co-expression profiles
• suppressor mutations
[Barkan Plant Physiol 155:1524]
High chlorophyll fluorescence (hcf)
mutants (maize and arabidopsis)
• Mutants in the nuclear genes required
for plastid biogenesis and function
• Many different genes!
• hcf/hcf > pale-green, yellow, or albino
seedlings; some fluoresce in the dark due
to dysfunctional photosystems
• hcf/hcf seedlings are lethal, but in
maize they grow large enough for
molecular analysis
wild-type seedling
wild-type seedling
mutant seedling
mutant seedling
White light
UV light
[Mutants of Maize, CSHLP]
Plant organelle RNA metabolism:
transcript processing & stability
Processed to di or mono-cistronic forms
Housekeeping endo-, 5’ exo- and 3’ exo-nucleases
Termini stabilized by stem-loops
Termini stabilized by PPR protein binding
Notice overlapping 5’ and 3’ termini
• Un-protected 3’ termini are de-stabilized by
polyA addition
similar to bacteria
in contrast to nuclear transcripts
•
•
•
•
[Barkan Plant Physiol 155:1524]
Pentatricopeptide repeat (PPR) proteins
Motif Structure of Arabidopsis PPR
Proteins
• Degenerate 35 amino acid repeats
• The number and order of repeat types
can vary in individual proteins
• The number of proteins falling into each
subgroup is shown
[Lurin et al. Plant Cell 16:2089]
Pentatricopeptide repeat (PPR)
proteins
• One of the largest multigene families in
plants
441 members in arabidopsis vs 7 in
humans
• Most are plastid- or mitochondriatargeted
• Site-specific RNA binding proteins
Modeled PPR motifs
& RNA ligand
(Fujii et al. Proc Natl Acad Sci U S A 108:1723)
Pentatricopeptide repeat (PPR)
proteins
Mediate most post-transcriptional RNA
metabolism
• Define transcript ends by nuclease
protection
• Intron splicing
• RNA editing
• Translation
Modes of action
• Recruit enzymatic protein complexes
• Simple site-specific binding
Protect from nucleases
Melt RNA 2o structures to allow
interaction with other processing,
splicing, translation & editing
factors
Plant organelle RNA metabolism:
intron splicing
Angiosperm mitochondria & plastid group II introns
• Characteristic 2o structure required for correct
splicing
• Evolutionary precursor to the nuclear splicosomal
RNAs
• Each intron requires a specific set of nuclearencoded splicing factors - mix & match
PPRs
CRM domain
Plant-specific
APO domain
RNA binding
PORR domain
protein families
}
[Barkan Plant Physiol 155:1524]
Plant organelle introns
Group I and Group II, defined by
characteristic secondary structures and
splicing mechanisms
[from Gillham 1994 Organelle Genes and Genomes]
Plant organelle intron splicing requires
multiple nuclear-encoded splicing factors
Evolutionary origin of splicing factor domains:
APO - eukaryotic, plant specific
CRM - eukaryotic, plant specific
CRS - prokaryotic peptidyl tRNA hydrolase
DUF794 - eukaryotic, plant specific
LAGLIDADG - prokaryotic group I intron maturase
PORR - eukaryotic, plant specific
PPR - eukaryotic, plant-expanded
RNaseIII - prokaryotic
WHY - eukaryotic, plant specific
[Watkins et al. Plant Cell 23:1082]
Organelle introns
How do we see whether introns are
spliced?
Reverse transcribe + PCR (RT-PCR)
DNA/
un-spliced RNA
<R 3’
cDNA 5’
<R 3’
PCR 5’ F>
spliced RNA
cDNA
PCR
5’
<R 3’
5’ F>
<R 3’
Others you may see:
• Ribonuclease protection
• RNA blot hybridization
• Organelle RNA seq
The maize crs1 and crs2 mutants
disrupt the splicing of different
group II introns
rps16
intron
[Jenkins et al. Plant Cell 9:283]
Group II intron trans-splicing
e.g. Chlamydomonas psaA transcripts
14 nuclear-encoded proteins & 1 additional plastid
transcript required
i1 5’ end
i1 3’ end
[Gillham 1994 Organelle Genes and Genomes]
Plant organelle RNA editing
Post transcriptional enzymatic conversion
of C > U, or less commonly, U > C
Given a fully sequenced organelle genome,
how would the RNA editing process be
detected?
genomic coding strand
5’ ....... ACG.....
unedited RNA
5’ ....... ACG.....
edited RNA
5’ ....... AUG....
edited cDNA
5’ ....... ATG.....
Occurs in plastids and plant mitochondria
• many more mitochondrial sites
Primarily in coding sequences
•conserves predicted protein
Creates initiation codons
ACG > AUG
Creates termination codons
CGA > UGA
Removes termination codons UGA > CGA
Changes amino acid coding
CCA > CUA
(P > L)
Silent edits
CTT > CTC
(L > L)
Plant organelle RNA editing
Edit sites within the same gene vary
among species
• An edit site in one species may be
“pre-edited” (correctly encoded in the
genomic sequence) of another species
• e.g. plastid psbL gene initiation
codon:
maize
ATGACA.....
tobacco ACGACA..... must be
edited to AUG (RNA) = ATG (cDNA)
for translation initiation codon
Evolution of plant organelle RNA editing
Not in algae
Observed in
every land plant
lineage except
Marchantiid
liverworts
[Knoop , Curr Genet 46:123]
RNA editing improves evolutionary
conservation
Table 1. Evolutionary conserved amino acid residues
created by C-to-U editing in ribosomal protein S12
(RPS12) of plant mitochondria
Amino acid residues encoded by unedited and edited
maize mitochondrial transcripts compared to amino
acid residues in RPS12 polypeptides from other taxa
[Mulligan and Maliga (1998) pp.153-161 In A look beyond
transcription
J Bailey-Serres and DR Gallie (eds) ASPB]
RNA editing by enzymatic de-amination
 32P UTP
V
 32P CTP >
32P CTP
[Rajasekhar and
Mulligan Plant Cell 5:1843]
[Russell, 1995, Genetics]
Short 5’ flanking sequences
define plant organelle RNA editing sites
[from Mulligan and Maliga (1998) pp.153-161 In A look
beyond transcription
J Bailey-Serres and DR Gallie (eds) ASPB]
RNA editing – genetic analysis
defines a trans-acting factor
[from Kotera et al. Nature 433:326]
RNA editing – genetic analysis
defines a trans-acting factor
The immunoblots implicating crr4 in NDH complex
biogenesis showed loss of the NDHH subunit, but
the affected RNA editing site is in the ndhD
transcript. What are some explanations for these
observations?
[from Kotera et al. Nature 433:326]
Genetic analysis defines a PPRmotif RNA editing factor
[from Kotera et al. Nature 433:326]
Translation of organelle genes
A significant regulatory process in
plastid gene expression
light-regulated chloroplast protein
accumulation increases 50-100 fold
w/out changes in mRNA accumulation
5’ untranslated region (UTR) is key
~ 1/2 of plastid transcripts have a 5’
Shine-Delgarno sequence (GGAG)
homologous to small subunit rRNA in
this region
Nuclear-encoded translation factors
bind 5’ untranslated region (UTR) (and
in some cases also the 3’ UTR)
message specific
activators of translation
Translation of organelle genes - PPR
protein RNA re-modeling enhances
ATPH translation
[Barkan Plant Physiol 155:1520]
Translation of organelle genes
(it’s not all about PPRs)
e.g. PSBA (D1 core of PSII)
Accumulation regulated by translation for
biogenesis and repair of the complex
• High D1 turnover due to light damage
Translation in Arabidopsis requires
•HCF173 discovered by forward genetics
•HCF244 discovered by in silico coexpression analysis
• Short-chain dehydrogenase superfamily
members w/out enzyme activity
Translation of organelle genes
HCF173 & HCF244 PSBA translation
factors in Arabidopsis
Mutant
phenotypes
Lack of D1 translation in each mutant
HCF173 is complexed with psbA RNA:
Slot blot of
HCF173-tHA
affinity tag antibody
captured RNAs from
wild-type and
HCF173-tHA
expressing line
[Schult et al. Plant Cell 19:1329; Link et al. Plant Physiol 160:2202]
Interdependence of organelle gene
expression processes - e.g. crp1
X
X
X
X
X
X
e.g. CRP1 - chloroplast RNA processing 1 mutant
• Which transcripts would be de-stabilized?
• What happens at the protein level and why?
[Barkan Plant Physiol 155:1524]
Interdependence of organelle gene
expression processes - e.g. crp1
Immunodetection of thylakoid membrane
protein accumulation
• Which proteins are reduced in abundance in
the crp1 mutant?
• Why are ALL of the PET protein subunits
missing? (Hint, there must be 50 ways to
lose a protein, name two!)
• Note PSAA and PSAB proteins, are also
slightly affected! (Hold this thought for
later.)
(Barkan et al. EMBOJ 13:3170)
Interdependence of organelle gene
expression processes - e.g. crp1
PET A,B,C,D protein translation studies
35S-labeled
leaf
proteins
immunoprecipitated
35S-labeled
in
organello
synthesized
proteins
immunoprecipitated
Which proteins are translated in the crp1
mutant? Which are not?
We saw PETA, B,C & D proteins did not
accumulate in this mutant.
What explains the difference between
translation and accumulation?
[Barkan et al. EMBOJ 13:3170]
Interdependence of organelle gene
expression processes - e.g. crp1
Protein
Translation in
crp1 mutant
Accumulation in
crp1 mutants
PETA
no
no
PETB
yes
no
PETC
yes
no
PETD
no
no
How does an RNA processing/stability
defect interfere with PET D translation?
How does failure to translate PETD
subunit interfere with the accumulation of
translated PETC?
How does failure to translate PETD
influence translation of PETA?
Interdependence of organelle gene
expression processes - e.g. CRP1
petB stop
codon
petD start
codon
RNA secondary structures
monocistronic petD
bi-cistronic
petB-petD
How does an RNA processing/stability
defect interfere with PETD translation
synthesis?
[Barkan et al. EMBOJ 13:3170]
Translation of organelle genes - PPR
protein RNA re-modeling enhances
ATPH translation
[Barkan Plant Physiol 155:1520]
How does failure to translate PETD
subunit interfere with the
accumulation of translated PETC?
Failure to assemble a protein complex >
degradation of unassembled subunits
Assembly dependent upon availability of all
subunits and co-factors
Plastids contain several proteases that are
homologues of bacterial proteases
Function in protein turn-over
Protease independent chaperone
functions (as seen in bacteria)
Bacterial – type proteases in plastids
Protease
Location and Function
in plastid
ClpP/ClpC
stroma
ATP-dependent serine
protease
degrades mis-targeted
proteins and cytb6/f
subunits
FtsH
stromal face of thylakoid
membranes
membrane-bound, ATPdependent metalloprotease
DegP
serine heat-shock
protease
degrades photo-damaged
PSII protein D1 from
stromal side
lumenal side of thylakoid
membranes
degrades photo-damaged
PSII protein D1 from
lumen side
Does failure to translate PETD
influence translation of PETA?
? Control by Epistasy of Synthesis (CES)
Regulation of protein synthesis by
absence of assembly partners
e.g. Down-regulation of tobacco nuclear
rbcS gene by antisense
•Decreased translation of rbcL in plastid
Not the case for failure to translate
PETA in crp1 mutants
CRP1 also associates w/ the 5’ region
of the petA transcript
• Immunoprecipitate CRP1 RNA-protein
complexes
• Slot-blot and hybridize
Immunoprecipitated RNA (pellet)
Unbound RNA (supernatant)
[Schmitz-Linneweber et al. Plant Cell 17:2791]
CRP1- RNA interactions
Why is the identification of two interaction
sites much more powerful than one?
consensus RNA binging site for CRP1 based
on two binding regions
D - model for
CRP1 protein –
RNA interaction
[Schmitz-Linneweber et al. Plant Cell 17:2791]
Retrograde regulation of nuclear
gene transcription through plastid
redox signals
PSA nuclear encoded subunit gene promoters
• Fused to GUS reporter gene
• GUS activity measured in response to light
changes
• How could we determine this is truly the
result of a plastid redox signal as opposed
to a nuclear light-regulated promoter?
[Pfannschmidt et al. J Biol Chem. 276:36125]
How are plastid signals transduced
to the nucleus?
[Pfannschmidt et al. Ann Bot 103:599]
Mitochondrial retrograde regulation
of nuclear genes
Plant mitochondrial respiratory electron transfer
chain includes an alternative pathway
• Single subunit alternative oxidase (AOX)
• Encoded by a nuclear gene (aox)
• Bypasses two of three sites for H+ transfer
coupled to ATP synthesis
• Transcription of nuclear aox is upregulated when
electron flow through the cytochrome pathway is
disrupted by the inhibitor antimycin A (AA)
Mitochondrial retrograde regulation
of nuclear genes
NtAI genes (Maxwell et al. Plant J 29:267)
• Nuclear genes up-regulated in response to AA,
including aox
• Seven additional genes identified by differential
mRNA display, most associated with stress
responses
acc oxidase (ethylene forming enzyme)
glutathione S transferase
Sar 8.2
cysteine protease
pathogen-induced lipase
SA-induced glucosyl transferase
Also induced by reactive oxygen species (ROS) (e.g.
H2O2)
Induction is blocked by antioxidants such as flavones
Of all inducers, AA is the most rapid implicating
mitochondria as a signaling source