biology and biotechnology of plastome engineering, presented at Shahid Beheshti University, Tehran, IRAN

1. ‫ﺑﺴﻢ ﺍﷲ ﺍﻟﺮﺣﻤﻦ‬
‫ﺍﻟﺮﺣﻴﻢ‬

2. Chloroplast Genome
Engineering
Biology and Biotechnology
Seyed Javad Davarpanah
Faculty of Bioscience, Shahid Beheshti University
June 19, 2010

3. Chloroplast genetic system
 A 50-290 kb double stranded circular molecule
 A pair of 20-30 kb inverted repeat (IR) sequence
 Prokaryotic protein synthesis machinery
 100 chloroplasts per mesophyll cell and 100 genome
copies per chloroplast (100 x 100 = 10,000 genome
copies per cell)

4. Chloroplast Genome Structure
Euglena
Pea
Typical Chloroplast Genome Exception

5. Higher plants plastome structure

6. Minicircle Structure in Dinoflagellates
Circular DNA molecules ranging in
size between 2.2-3.8 kb
Around 14 genes –Mostly one gene in a
minicircle (one gene - one circle or two
genes - one circle )
250-500 bp non-coding core region
Gene (s) always in the same orientation Typical coding minicircles
regarding the core region
Core region may function as replication
origin or promoter
Other Minicircles:
Empty, Chimeric minicircles, Jumbled minicircles and Microcircles

7. Nuclear transformation
 Biosafety
risk of gene flow to the environment
superweed production
pollen poisoning for non-target insects
 Stability of expression of transgene
transgene silencing (TGS) and (PTGS)

8. Chloroplast transformation
 Expression level of foreign genes is higher than nuclear
transformation; 5–80 (Chlamydomonas) or 500–10,000 (N
icotiana) DNA copies per cell
 Multiple genes can be introduced as an operon
 No risk of transgene escape – environmentally friendly
 No position effect
 No transgene silencing
 Sequestration of foreign proteins in the organelle

9. Advantages of transplastomic plants
 Transgenic pollen toxic to non-target insects of 60 major crop
plants, only 11 have no wild relatives
 No gene escape to WT (exceptions being alfalfa and possibly
rice and pea => No WT insensitiveness to herbicides
 Introgression of WT genes to transplastomic is in general in
unusual
 introgression of the common weed Raphanus raphanistrum
into Brassica napus (oilseed rape) occurred at higher rates
than the reciprocal cross of Brassica napus pollen into
Raphanus raphanistrum.

10. Stable transplastomic plants
• Transformation of plastids has already been achieved for
tobacco , Arabidopsis, soybean , cotton, lettuce, cauliflower,
poplar and potato
• The cereal crops rice, maize and wheat continue to be
recalcitrant
• plastid-mediated molecular pharming will lead to the
biofabrication of a range of biopolymers and pharmaceutical
proteins

11. Plastid transformed plants

12. Basics of Chloroplast Transformation
Chloroplast Transgenic Production Homologous Recombination Homoplasmy Process

13. Chloroplast transformation techniques
Biolistic delivery systems
Polyethylene glycol (PEG) treatment of protoplast
• For unknown reasons, the technique has a lower success rate
than biolistic bombardment
• long selection times required after initial DNA delivery
• technically demanding and requires specialized tissue culture
skills
Femtoinjection technique: injection of DNA material into
chloroplasts using syringes with extremely narrow tips
Agrobacterium-mediated plastid transformation:
• Two preliminary and thus far unconfirmed reports

14. Particle Delivery System

15. Advantages and disadvantages of biolistic
method
 Relatively high efficiency
 Technical simplicity
 Potential for mechanical shearing of large plasmids
during particle preparation or delivery
 Chemical attack by tungsten (a reactive transition metal)
which can promote modifications or cleavage of DNA

16. Advantages of femtoinjection technique
 Cells survive the injection
 Transformed cell can be spotted easily
 Cellular context remains intact
• The fate of the inserted gene or gene products to
be followed.

17. Galinstan Expansion Femtosyringe (GEF)
Chl autofluorescence GFP fluorescence overlay of both channels
marginal mesophyll cells of tobacco leaf
Ex: Phormidium laminosum, bla gene: spectinomycin
gfp gene under the control of a chloroplast rRNA promoter

18. Reporter gene strategies
• Gene coding for the green fluorescent protein (GFP)
• Resistance genes against lethal agents (e.g. spectinomycin and
streptomycin)
• disadvantage of resistance marker genes: transformed cells must be
traced by stringent methods
• Vectors carrying the bacterial gene aphA-6, coding for an
aminoglycoside phosphotransferase that detoxifies kanamycin or
amikacin
• FLARE-S system, the aminoglycoside 3′′ adenyltransferase (aadA
gene), which confers resistance against spectomycin and
streptomycin,is translationally fused to the gfp gene of Aequorea
victoria
• In the case of an optical marker like GFP, difficulties arise with the
regeneration of a plant from a single GFP-expressing cell
Reporter gene strategy: genetic contamination problems

19. Reasons to produce marker-free
transplastomic plants
 Potential metabolic burden imposed by high levels of
marker gene expression
homoplastomic state :the marker gene product 5% to 18% of
the total cellular soluble protein
 Shortage of primary plastid selective markers
only genes that confer resistance to spectinomycin and
streptomycin (aadA) or kanamycin (neo or kan and aphA-6
 Opposition to having any unnecessary DNA in
transgenic crops, especially antibiotic resistance
genes

20. Approaches for production of marker free
transplastomic plants
Homology-based excision via directly repeated
sequences
Excision by phage site-specific recombinanses
Transient co-integration of the marker gene
Cotransformation-segregation approach

21. Homology-based excision of Marker gene
via directly repeated sequences
Recognition sequence of site-specific recombinanse

22. Marker gene excision by phage site-
specific recombinanses

23. Marker gene excision by phage site-
specific recombinanses
1-transplastomics carry marker gene flanked by two
directly oriented recombinase target sites
2-removal of marker gene by introduction of a gene
encoding a plastid-targeted recombinase in the plant
nucleus
• recombinases (Cre and Int)
• absence of homology between the attB and attP sites
and the absence of pseudo-att sites in ptDNA=> Int
better than Cre

24. Transient cointegration of the marker gene
to obtain marker-free plants

25. Cotransformation-segregation

26. New marker genes applying RNA editing
in plastids
 conversion of specific C nucleotides to U in plastids
 Mediated by a nuclear encoded complex
 Some plastid genes (e.g., psbL, ndhD, rpl2) the start codon is
encoded as ACG and must be edited to AUG
 =>constructing new selectable marker gene only expressible
when integrated into the plastome

27. Comparison of Systems for Production of Heterologous Protein
System Overall Production scale-up Product Glycosylation Contamination Storage cost
cost timescale capacity quality risks
Bacteria Low Short High Low None Endotoxins Moderate
Yeast Medium Medium High Medium Incorrect Low risk Moderate
Mammalian High Long Very low Very Correct Viruses, prions Expensive
cell culture high and oncogenic
DNA
Transgenic High Very long Low Very Correct Viruses, prions Expensive
animals high and oncogenic
DNA
Plant cell Medium Medium Medium High Minor Low risk Moderate
cultures differences
Transgenic Very low Long Very High Minor Low risk Inexpensive
plants high differences

28. Heterologous genes expressed stably in plastids of tobacco

29. Production of various protein classes
• expression of genes coding for insecticidal proteins or
allowing for herbicide resistance
Bacillus thuringiensis (Bt) toxin: the gene (cry1A)
coding for the Bt toxin Cry1A(c)
cry2Aa2 Bt gene
Nucleus: suboptimal production of toxin=> toxin
resistance
Chloroplast:100% mortality of resistant insects
20-30 fold higher Bt prototoxin production

30. Oxyfluorfen resistance
• plastomic insertion of the Bacillus subtilis gene
encoding protoporphyrinogen oxidase (protox)
• a diphenyl herbicide resistant
• higher degree of oxyfluorfen resistance

31. Glyphosate resistance
• EPSPS: a nuclear encoded, plastid targeted enzyme
• Integration of the petunia EPSPS (5-enol-pyruvyl
shikimate-3-phosphate synthase) gene into the
tobacco plastome
• Overproduction of EPSPS
• Glyphosate resistance

32. • production of a human somatropin in a soluble biologically
active form
• biodegradable protein-based polymers in tobacco
• introduce into plants a set of bacterial genes for the
biosynthesis of polyhydroxyalkanoates (PHAs)
• PHAs: a class of biodegradable polymers
• fermentative production has proven too costly for large-scale
production
• Targeting of PHA biosynthetic genes from Ralstonia eutropha
• Proteins involved in the metabolic pathways of plastids
Rubisco, Reaction Center proteins
rbcL of Synechococcus: mRNA production but no protein or
enzyme activity

33. Engineering of plastid metabolism
 Site-directed mutagenesis of Rubisco
o deletion of rbcL, replacement with chimeric plastid targeted LSU
o rbcL replacement with cyanobacterial
homologues: no translation
 Plastid reverse genetics
• function of several chloroplastic open reading frames (ORFs)
ycf1,ycf2,ycf9 transplastomics: all lines heteroplasmic
ycf9 ORF: stabilisation of LHC
ycf6: involved in construction of cyt b6f complex
• functioning of plastidic RNA
functioning of plastidic RNA endonuclease
• chloroplast structure and physiology only partly suffered from knocking
out plastid-encoded RNA polymerase

34. Requirements for widespread application of
chloroplast engineering
the number of plant species to which plastome
technology is applicable needs to be increased
considerably
the success rate of gene insertion into the plastome
has to be increased
the screening protocols must be simplified and
become applicable to a large range of plant species

35. Thanks for your patience