For a greener future

Engineering the Plastid

Sachin S RawatSchool of Biotech, GGS IP University

A Look at the Plastid

A Plastid is a….

Major organelle of plant and algal cellsSite of manufacture and storage of

important chemical compoundsHas circular, dsDNA copiesReplicates autonomously of the cellThought to have been originated from

endosymbiotic bacteria

Plastid genes show maternal inheritance

Derived from proplastids in meristem

Have diverse functions

Chloroplasts – green plastids – for photosynthesis

Chromoplasts – coloured plastids – for pigment synthesis and storage

Gerontoplasts – control dismantling of photosynthetic apparatus during senescence

Leucoplasts – colourless plastids – monoterpene synthesis

Leucoplasts include amyloplasts (starch), elaioplasts (fats), proteinoplasts (proteins) and tannosomes (tannins)

120-130 plastid genes

Are densely packed and fall into 2 categories:

Photosynthesis-related genesGenetic system genes - genes for rRNAs,

tRNAs, ribosomal proteins and RNA polymerase subunits

A Fresher Look at the Plastid

Why plastid transformation?

High protein expression levels

Absence of epigenetic effects

Uniparental inheritance is commercially favoured

Easy transgene stacking in operons

Increased biosafety – Since plastids are maternally inherited, they aren’t transmitted by pollen

Hurdles to ‘transplastomic’ plants

Difficulty in delivering foreign DNA through double membrane of the plastid

The enormous copy number (polyploidy) of the plastid genome

The desired genetic modification must be in each copy of plastid genome in each cell

Failure to achieve homoplasmy results in rapid somatic segregation and genetic instability

Repeated rounds of selection and regeneration are required

DNA delivery into plastids2 successful methods include biolistics and

polyethylene glycol-mediated transferBiolistics is preferred as it is less time-

consuming and demanding

Integration of foreign DNA into plastid genome occurs via homologous recombination

Homologous recombination operates in plastids at a high efficiency

Biolistic chloroplast transformation and transgene integration into theplastid genome via homologous recombination

Recent success Expression of Bt toxin gene from the tobacco

plastid genomeHigh accumulation levels of Bt toxin protein (3-5 %

of TSP)Plants with high-level resistance to herbivorous

insects

Co-expression with upstream ORFs further increased Bt toxin accumulation and even resulted in its crystallization in chloroplast

Production of somatotropin (7% TSP) in tobacco plastids

Case Study I – Lactuca sativa

Protoplast isolationLettuce seeds were sterilized and sown on

MS medium with 2% sucroseShoot tips from leaves obtained were

transferred to MS medium with 3% sucroseThe leaves were cut into pieces and

incubated in PG solution, followed by enzyme solution consisting of 1% cellulase and .25% macerozyme

Protoplast suspension was filtered through nylon mesh

Protoplasts were collected at surface after centrifugation at 70g for 8min

Transformation and culture

10µl transforming DNA and 0.6ml PEG solution was added to protoplast suspension and incubated at 25ºC for 10min

Protoplasts were mixed with 1:1 solution of B5 and 2% agarose to a density of 3.6 X 104 protoplasts per ml

The suspension was plated onto Petri dishes and cultured at 25ºC in the dark

Selection was initiated on the 7th day by fresh medium containing spectinomycin dihydrochloride

AnalysesPCR – specific primers were used to assess

the presence of aadA gene in resistant cell lines

Immunoblot analysis – using HRP-conjugated secondary antibodies

Southern and Northern blots were performed to look for target genes and their transcripts

After 2 weeks, non-transformants were yellow while spectinomycin-resistant seedlings were green and growing vigorously

100% of spectinomycin-resistant lettuce cell lines were true plastid transformants

A limitation was the high frequency of polyploid cell lines

Production of humantherapeutic proteins

Why lettuce is favoured over tobacco? Most of the plant is leaf tissue and this tissue

contains the greatest number of plastids per cell

Unlike tobacco, lettuce has no toxic alkaloids that need to be removed - low purification and downstream processing costs

Lettuce is a relevant human foodstuff that can be consumed without cooking

Case Study II – Petunia hybrida

Plastid transformation

Leaf pieces were placed on MS medium supplemented with 1 mg/l 6-benzylaminopurine, 0.1 mg/l IAA, 30 g/l sucrose and 0.8% agar (MSB30)

Leaves were bombarded with 1µm, vector-coated gold particles from distance of 6cm

Incubated in dim light for 48h at 25ºCLeaves were transferred to MSB30 medium

with 200mg/l each of streptomycin sulfate and spectinomycin dihydrochloride pentahydrate

Resistant shoots first appeared after 8 weeks

Vector design

AnalysesDNA blot – gene specific primers were usedGUS assay – 5-Bromo-4-chloro-3-

indolylbeta-D-glucuronic acid was used to compare the protein expression levels between the wild type and the transformants by detecting fluorescence

Selection on two antibiotics overcomes the problem of spontaneous resistant mutants associated with using spectinomycin alone

Comparing plastid transformants with non-transformants

Good model to study plastid biology

N. tabacum is amphi-diploidA. thaliana doesn’t give rise to fertile

transplastomes

These limitations are overcome in Petunia as:

P. hybrida is diploidSuitable for mutation screening to identify

nuclear loci affecting the maintenance and expression of plastid transgenes

A Look at the Future

Metabolic pathways into plastids?

Cost-effective production platform for pharmaceuticals and nutraceuticals

Production of trehalose in tobacco chloroplasts

Tryptophan overproduction by feedback-insensitive synthesis of α-subunit of anthranilate synthase

Simplifying technology, extending crop range

Can we engineer photosynthesis?

Efficiency of photosynthesis

The most abundant protein in the world

Its CO2:O2 specificity that matters

Getting a better RubisCO for your plant

Equally precise tools for nuclear genome required

Plastids for Synthetic Biology

A compact, minimal genome

High transgene expression and low cost ideal for synthetic biology

Designing totally new plastids

References

Bock and Khan; Taming plastids for a green future; Trends in Biotechnology

Lelivelt et al.; Stable plastid transformation in lettuce; Plant Molecular Biology

Zuilen et al.; Stable transformation of Petunia plastids; Transgenic Research

Thank you