Regulation of Chloroplast Gene
Expression
• Studied principally during photomorphogenesis
– i.e., development of cotyledons and leaves
during "greening" (etioplast -> chloroplast).
• Also studied (in mature chloroplasts) during
light-dark cycles, and in response to certain
stresses (heat, cold, radiation).
• Multiple levels are regulated for most genes.
• Difficult to generalize, but some trends emerge.
Plastid Transcriptional Regulation
•
Transcriptional regulation is often global or
large-scale
– NEP functions early in development, PEP
dominates later (etioplast  chloroplast)
– PEP-transcribed genes increase or decrease
together
•
•
E.g. - overall transcription increases during "greening",
but decreases during chloroplast  chromoplast
There are examples of gene-specific
transcriptional regulation
– psbD/psbC promoter switching in response to light
Plastid development is plastic, mostly under
nuclear control.
Shoots:
NEP
proplastids
PEP
light
etioplasts
chloroplasts
Declines
chromoplasts
Roots: More NEP, less PEP
proplastids
amyloplasts
Need to express accD , and ycf1 and ycf2 in all
organs/tissues, essential for growth.
psbD-psbC Light-responsive
Promoter (LRP or BLRP)
●
Preferentially utilized in the light (not dark); stimulated
by blue and UV light.
●
Also shows circadian rhythm of utilization.
●
Evolutionarily conserved among higher plants.
●
PEP-type promoter, but the -35 region not necessary.
●
●
2 upstream regions important for the light-response:
PGT and AAG boxes.
Boxes bind proteins (PTF1, AGF); binding of PTF1 is
inhibited by ADP-dependent phosphorylation (ADP
levels increase in darkness).
BLRP
promoter
psbD BLRP
Schematic diagram of the barley psbD-LRP and constructs used for plastid transformation. A. The boxed regions identify conserved sequences which include
the PGT-box (−71 to −100), AAG-box (−36 to −56) and the prokaryotic-like −10 (−7 to −12) and −35 (−28 to −33) promoter elements. The psbD open-reading
frame is shown at the far right. The direction of transcription is represented as an arrow and the initiation site is labeled as +1. A sequence alignment between
the barley (Sexton et al., 1990b) and tobacco (Shinozaki et al., 1986) psbD-LRP was made with the ClustalW 1.7 Multiple Sequence Alignment Program. Aligned
nucleotide sequences corresponding to conserved sequences are boxed in and labeled accordingly. Numbering of nucleotides and designation of conserved
promoter elements are in accordance with the structure of the barley psbD-LRP from the transcription initiation site (+1).
(From Thum et al. 2001)
Models of transcription complexes associated with the psbD BLRP, rbcL and psbA
promoters
- extra TATA box
likely maintains high
rate of transcription
in mature chloroplast
Kim, M. et al. J. Biol. Chem. 1999;274:4684-4692
Regulation of RNA splicing & stability
1. Splicing of psbA introns (Group I) in Chlamy
is strongly promoted by light (& redox).
2. Splicing of some photosynthetic genes’
introns (Group II) is inefficient in maize
roots (amyloplasts), but efficient in leaves.
3. Stability of some plastid mRNAs increases
during greening (psbA), but most
decrease in mature chloroplasts in the
light.
Light-Dependent
Splicing of psbA
psbAi2 intron
LSU rRNA intron
Translational Regulation
•
•
Cp mRNAs are relatively long-lived (half lives of
0.5 to 8 h or more)
Translation is regulated by:
1. Global changes in rate (e.g., light-dark
cycles)
e.g. - high in daytime, low at night
2. Preferential translation of specific mRNAs
under certain conditions.
e.g.- very high light intensity increases
psbA translation and decreases rbcL
translation
Light-activated translation of psbA mRNA
• Complex of proteins that
bind to the 5’ UTR of
psbA mRNA in the light.
• Demonstrate with gelshift (electrophoretic
mobility shift) assay.
Lane 1 – control (no protein extract)
Lane 2 - extract from light-grown cells
Lane 3 - extract from dark-grown cells
S. Mayfield lab
Box 9.4 in Buchanan et al.
Proteins in complex that bind to the
5’ UTR of psbA mRNA
1. PABP - similar to a polyA-binding protein,
binds A-U rich region in the 5’ UTR,
activates translation
2. PDI - a protein disulfide isomerase (reduces
disulfide bonds on certain proteins),
activates PABP to bind RNA
3. Kinase – responds to ADP levels, at high
[ADP], kinase deactivates PDI by
phosphorylating it
Model for Activation of psbA translation by Light
via photosynthesis.
Fig. 9.23 in Buchanan et al.
Ribulose-1,5-bisphosphate carboxylase/oxygenase,
RuBPCase (or Rubisco)
• Catalyzes carboxylation of ribulose-1,5
bisphosphate: CO2 + RuBP  3PGA (x 2)
• 2 subunits, large (LS) and small (SS)
– 8 copies of each per holoenzyme
• LS gene (rbcL) in the chloroplast
• SS gene (rbcS) in the nucleus
• extremely abundant, because inefficient
– Pyrenoid in algae is mostly RuBPCase
• regulated during light-dark cycles
– enzyme more active in the light
– also synthesized mainly in the light
Translational regulation of RuBPCase LS by SS
Incoming SS somehow promotes translation of rbcL mRNA!
Bogorad
lab
Fig. 9.16 in Buchanan et al.
There also seems to be autoregulation of rbcL translation:
Cohen et al. (2006) Plant Physiol. 141, 1089; Wostrikoff and Stern (2007) PNAS 104, 6466
Rough Thylakoids
• Polyribosome (polysomes) can be observed
bound to thylakoid membranes.
• At least some of these polysomes are
attached to the membrane by the nascent
(“new”) protein.
• Suggests these polysomes make thylakoid
membrane proteins and simultaneously
insert them into the membrane.
• Chloroplasts also contain a Signal
Recognition Particle (SRP) homologue.
Thylakoid-bound polysomes from Chlamydomonas
Occur in the light period of a light-dark cycle
polysome
polysome
A. Michaels, M. Margulies and G. Palade (~1972)
Stabilization
of nascent
chlorophyll binding
proteins of
PSI and PSII
with
Chlorophyll
Fig 9.24 in Buchanan et al.
Regulation of protein stability in chloroplasts
Protein stability is regulated by:
1. binding of cofactors (e.g., chlorophyll and
carotenoids)
2. assembly with other subunits in a multi-subunit
enzyme complex (PSI,PSII, ATP syn)
ATP Synthetase
Photoinhibition: inhibition of
photosynthesis at very high light flux
Photosystem II damage is critical.
Box 9.6 in Buchanan et al.
psbA encodes ~32-35 kDa D1 polypeptide of PSII
Yamamoto, Plant Cell Physiol. 2001
D1 protein turns over rapidly because it becomes damaged
in the light.
D1 turnover and replacement is
ongoing and critical
• At photoinhibitory light intensity, D1
protein of PSII is damaged faster that it
can be removed (and degraded).
• At most lower light intensities,
degradation, synthesis and replacement
of D1 keeps up with the damage rate.
Retrograde Signaling & Regulation
Retrograde Regulation
- Regulation of nuclear genes by
the chloroplast
- Nuclear genes typically
encode chloroplast proteins
- Signaled by:
(1) Developmental state of the
plastid/gene expression
(2) Photo-oxidative stress
Anterograde Regulation
- Regulation of chloroplast genes
by nuclear gene products
- Occurs at most levels of
expression
GUN Genes and Retrograde Signaling
Zhang,D (2007)
Signaling to the nucleus
w/a loaded GUN.
Science 316, 700-701.