Regulation of Chloroplast Gene Expression Studied principally during photomorphogenesis – i.e.,...

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: light

proplastids etioplasts chloroplasts

chromoplasts

Roots:proplastids amyloplasts

NEPPEP

Declines

More NEP, less PEP

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).

BLRPpromoter

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)

psbD BLRP

Kim, M. et al. J. Biol. Chem. 1999;274:4684-4692

Models of transcription complexes associated with the psbD BLRP, rbcL and psbA promoters

- extra TATA boxlikely maintains high rate of transcription in mature chloroplast

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.

psbAi2 intron

LSU rRNA intron

Light-Dependent Splicing of psbA

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 gel-shift (electrophoretic mobility shift) assay.

Lane 1 – control (no protein extract)Lane 2 - extract from light-grown cellsLane 3 - extract from dark-grown cells

Box 9.4 in Buchanan et al.

S. Mayfield lab

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!

Fig. 9.16 in Buchanan et al.

Bogorad lab

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

A. Michaels, M. Margulies and G. Palade (~1972)

polysome

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

Box 9.6 in Buchanan et al.

Photosystem II damage is critical.

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.