Chloroplast transformation
Plastids contain DNA – plastome
- Maternal inheritance
(advantage for biotechnological application)
(Mirabilis japonica, Correns 1909)
- 100 x 100 plastoms/cell
- Prokaryotic origin
- gene transfer
Gene expression in plastids is procaryotic
Inheritance in plastids
- Pelargonium:
- biparental
- maternal (most of the angisperms)
- paternal (gymnosperms, Sequoia, Pinus)
plastome
- DNA is attached to thylakoid membrane (nucleoid)
- 15 nucleoids/plastid, 10 DNA molecules/nucleotid (polyploid)
- circular DNA
- 130 bis 160 kb
- inverse duplication
- small and large single copy region
- loss of inverse duplication e.g. conifers, Papilionaceae
Epiphagus
The plastome of the holoparasiteEpifagus virginiana issubstantially reduced
Model system for plastidgenetics
Plastomes of land plants
Genes of the plastome
Gene expression in plastids requires pro- and eukaryotic elements
Operons und Introns
Most of the promoters are procaryotic – but not all of them
Plastids contain two RNA-polymerases
- Epiphagus: lost the genes for RNA-polymerases, but still contain white plastids
- nuclear-encoded RNA-polymerase - plastid-encoded RNA-polymerase
- phage type - bacterial type- one subunit - ~13 subunits, nuclear- and plastid-encoded
- sigma factors (bacteria-like) - Expression of early genes - Expression of late genes
psbB operon: complex processing steps
psbB operon- multiple promotors, multiple transcription start sites
- both strands encode genes
- polycistronic transcripts
- primary transcript is large and unstable
- RNA codes for independent proteins
- transcript ripening, oligocistronic transcripts
- monocistronic transcripts
- specific endonucleases
- Exonucleases: processing of 3´-ends
- hair pin loops stabilizes RNA
- secondary structures prevent degradation
Editing change plastid transcripts –Hydrolytic deamination of cytidine to uridine
Most of the transcripts are stable
Many genes from plastids were transferred to thenucleus
- DNA fragment
- as RNA after reverse transcription (e.g. as edited transcripts)
Chloroplasts transformation
Most of the transformationprotocols use protoplasts
Regeneration is similar to nucleartransformation
Pt transformation works in monocots - rice
Problem: generation of homoplastomic lines
Plastid transformationvectors are based on
homologousrecombination
eventsI
Different from nucleartransformation
Plastid transformationvectors are based on
homologousrecombination events
II
Careful choice of insertion site
Requirement of flanking sequence for
recombination
aad as selection marker- use of two selectable markers
- aad: procaryotic expression
- neomycin: eukaryoticexpression
Reciprocal crosses show maternalinheritance (here: resistance gene)
Advantages of plastidtransformation I
• Huge production of proteins• Maternal inheritance• Application of eatable plastids:
– Chromoplasts from tomato (no denaturation)– Amyloplasts from potato (boiling)
Advantages of plastidtransformation II
• Chloroplast gene expression is mainlyregulated posttranscriptionally
• mRNA is present, although proteinsdo not accumulate (photosynthesisgenes in tomato and potato)
• Transformation of chloroplasts, expression in etio- or amyloplasts
Expression can be >500-fold higherthan in the nucleus
Gene inactivation identifies the role of plastidencoded proteins: inactivation of plastid RNA
polymerase
Tomato and potato are crucialplants for plastid transformation
Higher carotenoid level