chloroplast transformation · plastome - dna is attached to thylakoid membrane (nucleoid) - 15...

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