Plastids

Plastids (derived from proplastids)

1. Chromoplast2. Chloroplast3. Amyloplast4. Leucoplast5. Elaioplast6. Etioplast

In plants, meristamatic cells contain 10-14 proplastids, each carrying 1-2 nucleoids per proplastid, whereas leaf cells may contain 100 chloroplasts, with 10-14 nucleoids each. There are several ptDNA per nucleoid. Thus proplastids contain lower copies of ptDNA than chloroplasts.

Plastidnucleoid

ptDNA copies

The Structure of of O. sativa Chloroplast Genome

~120 genes~50 transcription units

Two inverted repeatsOne large single copy regionOne small single copy region

These are salient features of any higher plant plastids

Conserved Features of Chloroplast Genomes in Higher Plants

LSC IRB SSC IRA

86,686 25,341 18,571 25,341

82,355 22,748 22,74812,536

80,592 20,799 20,79912,334

81,095 10,058 10,058 19,813

65,696 495 49553,021

19,799 22,735 22,7354759

Tobacco (155,939 bp)

Maize (140,387 bp)

Rice (134,525 bp)

Marchantia (121,024 bp)

Black pine (119,707 bp)

Epifagus (70,028)

LSC= Large single copy region SSC= small single copy region

Genes Encoded in the Chloroplast Genomes in Higher Plants

Gene Designation Gene Product I. Genetic System Chloroplast RNA genes

rDNA Ribosomal RNAs (16S, 23S, 4.5S, 5S)trn Transfer RNAs (30 species)

Gene transcriptionrpoA, B, C RNA polymerase , , ’ subunitsssb ssDNA-binding protein

Protein synthesisrps2,3,4,7,8,11 30S ribosomal proteins (CS) 2, 3, 4, 7, 8, 11rps12, 14, 15, 16, 18, 19 CS12, 14, 16, 18, 19rpl2, 14, 16, 20, 22 50S ribosomal proteins (CL) 2, 14, 16, 20, 22infA Initiation factor I

II. Photosynthesis Photosynthetic proteins

rbcL RUBISCO large subunitatpA, B, E ATP synthetase CF1, , subunitsatpF, H, I ATP synthetase CF0I, III, IV subunitspsaA, B, C Photosystem I A1, A2, 9-kDa proteinpsbA, B, C, D, E Photosystem II D1, 51 kDa, 44 kDa, D2, Cytb559-

9kDapsbF, G, H, I Photosystem II Cytb559-4kDa, G, 10Pi, I proteinspetA, B, D Electron transport Cytf, Cytb6, IV subunits

Respiratory proteinsndhA, B, C, D NADH dehydrogenase (ND) subunits 1, 2, 3, 4ndhE, F NDL4L, 5

III. Others Maturase matK

Protease clpPEnvelope membrane protein cemA

Organization of chloroplast genes into operons

psbB

psbT psbH

petB

Intron

Intron

petD

psbN

Polycistronic mRNA

Monocistronic mRNA

The Endosymbiont Theory

Common ancestor of plastidand modern cyanobacteria

Common ancestor of mitochondiraand -group of modern

proteobacteria

Protoeukaryotic cell

Photosynthetic eukaryotic cell

Flowering plant

Photosynthetic C-reductionRespiration

The Endosymbiont Theory

Supporting Evidences

1. Molecular architecture and genome replication a) Plastid genomes are naked covalently closed circular DNA molecules (devoid of

histones).

b) Replication of plastid DNA is independent of the nuclear genome replication

c) Promoters of most chloroplast genes contain DNA sequences similar to the E. coli ‘-10’ and ‘-35’ promoter motifs.

d) Chloroplast open reading frames are polycistronic.

e) Plastid genomes contain few moderately or highly repetitive sequences

f) Chloroplast genomes of Euglena, Chlamydomonas and most angiosperms carry 2 or 3 rRNA genes which are similar in size to their prokaryotic homologs (23S, 16S,

5S)

Chloroplast promoters

psbA TTGGTTGACATGGCTATATAAGTCATGTTATACTGTTCAAT

psbA TTGGTTGACACGGGCATATAAGGCATGTTATACTGTTGAAT

rbcL TGGGTTGCGCCATATATATGAAAGAGTATACAATAATGATG

atpB TCTTGACAGTGGTATATGTTGTATATGTATATCCTAGATGT

trnM TTATATTGCTTATATATAATATTTGATTTATAATCAATCTA

Mustard

Spinach

“-35” “-10”

1) Chloroplast promoters- similar to bacterial minimal promoter.

psbA TTGGTTGACATGGCTATATAAGTCATGTTATACTGTTCAAT

psbA TTGGTTGACACGGGCATATAAGGCATGTTATACTGTTGAAT

rbcL TGGGTTGCGCCATATATATGAAAGAGTATACAATAATGATG

atpB TCTTGACAGTGGTATATGTTGTATATGTATATCCTAGATGT

trnM TTATATTGCTTATATATAATATTTGATTTATAATCAATCTA

Mustard

Spinach

“-35” “-10”

Features of chloroplast transcription

2) Polycistronic.

3) Cis-elements located in the 5’-UTR.

4) Nuclear-encoded transcription factors.

Features of chloroplast translation (similar to prokaryotic translation)

1) Makes use of 70S ribosomes.

2) Uses fMet-initiator tRNA for the translation initiation codon.

3) The mRNAs are not capped.

4) The mRNAs are not poly-adenylated.

5) Ribosome binding occur in Shine-Delgarno-like sequence motif in the 5’-UT of mRNA.

6) Not coupled to transcription and trnaslational units can occur as stable ribonucleoprotein complexes.

The Endosymbiont Theory (cont.)

Supporting Evidences

2) Transcriptiona) RNA polymerases from cyanobacteria (e.g. Chlamydomonas) and higher plants (e.g. maize) are more similar to the eubacterial than to the nuclear homologs.

b) Genes encoding for proteins of related functions are organized into operons and thus are co-transcribed.

c) The limiting regulatory step of gene expression is at post-transcriptional and translational level.

d) Transcription terminators are more similar to bacterial sequences.

d) A minor fraction of chloroplast mRNAs are polyadenylated.

The Endosymbiont Theory (cont.)

Supporting Evidences

3) Translation

a) Plastid ribosomes are more similar to prokaryotic ribosomes than to their cytoplasmic counterparts: cytoplasmic ribosomes- 80S (40S + 60S subunits)

Plastid and prokaryotic ribosomes- 70S (30S + 50S subunits) Antibodies raised against 70S and 30S

subunits of plastid ribosomes are active against E. coli

b) Plastid ribosomal RNA gene sequences are more similar to modern cyanobacteria (e.g. Synechococcus lividus) than to their nuclear counterparts.

The Endosymbiont Theory (cont.)

Supporting Evidences

4) Others (biflagellate protists)The case of Cyanophora paradoxa (and other types of marine nudibrachs or sea slugs)

EndosymbioticCyanobacterium

PhotosyntheticCyanelle

Cyanophora paradoxa

Plastid transformation

Basic Requirements:1) Method of delivery (Biolistic method)

2) Selectable marker (dominant marker)

Firing pin

Helium gasNylon macro-projectile

Micro-projectileDNA-coated gold particles

Vents

Plate to stop nylon projectile

Target cellsor tissues

3”-adenylyltransferase (aadA)

Spectinomycin inhibits protein biosynthesis (70S ribosomes)

Spectinomycin Adenylylspectinomycin (inactive protein synthesis inhibitor)

AMP

Construct:

“-35” “-10” 5’-UTR SD aadA-ORF 3’-UTR

First successful plastid transformation was reported in 1988 for chlamydomonas. Then in 1990 for tobacco. Since then only tomato has been added to the list of reproducible systems, though reports exist for cotton, wheat etc.

A transformed plastid genome is formed bytwo recombination events that are targeted byhomologous sequences. The plastid genomesegments that are included in the vector aremarked as the left (LTR) and right targetingregions (RTR).

Chloroplast Genetic EngineeringChloroplast Genetic EngineeringProkaryotic – No need for codon optimization10, 000 copies per cell – high expressionMaternal inheritanceNot expressed in fruits ?Multigene engineering

Homologous recombination

Advantages of Chloroplast Advantages of Chloroplast TransformationTransformation

Gene Gene ContainmentContainment

MaternalMaternalInheritanceInheritance

No GeneNo GeneSilencingSilencing

No PositionNo PositionEffectEffect

Hyper-Hyper-expressionexpression

MultigeneMultigeneEngineeringEngineering

No VectorNo VectorSequencesSequences

NoNoPleiotropicPleiotropic

EffectsEffects

Cry2Aa2 Single Gene Expression

00.050.1

0.150.2

0.250.3

0.350.4

Control young mature oldTransgenic Leaf Age

% T

ota

l So

lub

le P

rote

in

Cry2Aa2 Operon Expression

01020

304050

Control young mature oldTransgenic Leaf Age%

To

tal S

olu

ble

Pro

tein

A.

B.

100 fold higher expression obtained by plastid transformation (B) compared to nuclear transformation (A)

Accelerated gold particlecoated with transforming DNA

~10,000 plastidgenomes/cell

Primary plastidtransformation event (change

ofsingle plastid

DNAmolecule)

Cell and organelle

divisions under antibiotic selection

(heteroplasmy)

Several cycles of

antibiotic selection

(homoplasmy)

Heteroplasmy vs homoplasmy

nucleus

chloroplast proplastid

sorting

biogenesis

 Selection of transplastomic clones by spectinomycin resistance. (A) Spectinomycin inhibits callus formation, greening, and shoot regeneration from tobacco leaf segments on shoot regeneration medium. Transplastomic clones are resistant to spectinomycin and are identified as green shoots or calli. (B) The shoots are chimeric, visualized by accumulation of green fluorescent protein in transplastomic sectors. Spectinomycin resistance is not cell autonomous as sensitive sectors are also green. (C) Spontaneous spectinomycin resistant mutants are sensitive (top), transplastomic clones are resistant to streptomycin (bottom) when cultured on a selective streptomycin (500 mg/L) medium.

Comparison of the nuclear and plastid genomes of angiosperms

Nuclear genome Plastid genome

Chromosomes Two copies of each of ~60 copies of a single circularmany chromosomes; chromosome per plastidthe number of ~50–60 chloroplasts per cellchromosomes per diploid cell is species-specific

Genes per chromosome Could be thousands ~120–150

Arrangement and Each gene is separate Many genes are in operons transcription of genes (individually transcribed ) (transcribed together)

Currently known primary markers are resistance to spectinomycin, streptomycin, and kanamycin, which inhibit protein synthesis on prokaryotic-type plastid ribosomes. These antibiotics inhibit greening, cell division, and shoot formation in tobacco culture. Therefore, greening, faster proliferation, and shoot formation were used to identify transplastomic clones on a selective medium. The first transplastomic clones were obtained by spectinomycin selection. Because spectinomycin allows slow proliferation of nontransformed tobacco cells it was assumed that the choice of a drug that enables such "nonlethal" selection is important to recover transplastomic clones. However, transplastomic clones were soon identified by kanamycin selection using an antibiotic concentration that is considered "lethal" (50 mg/L). Thus, slow proliferation of nontransformed cells on a selective medium is not an essential feature of the selection scheme. Initial transformation vectors carried a plastid 16S rRNA (rrn16) gene with point mutations that prevent binding of spectinomycin or streptomycin to the 16S rRNA. The rrn16 target site mutations are recessive, and were 100-fold less efficient than the currently used dominant aadA gene. Streptomycin resistance encoded in the rps12 ribosomal protein gene was also included in an early vector. The neo (aph(3')IIa) gene encodes neomycin phosphotransferase II [NPTII; APH(3')-II], and was used to select transplastomic clones in tobacco. The aphA-6 gene encodes aminoglycoside phosphotransferase or APH(3')-VI, and was used to select transplastomic clones by kanamycin and amikamycin resistance in Chlamydomonas and by kanamycin resistance in tobacco. Direct selection for spectinomycin resistance and for highly expressed kanamycin resistance genes, on average, yield one transplastomic line in a bombarded leaf sample.

Selection markers

Most of the lecture material is derived from:

1. Pal Maliga (2004) PLASTID TRANSFORMATION IN HIGHER PLANTS. Annual Review of Plant Biology. 55: 289-313.

2. Pal Maliga (2002) Engineering the plastid genome of higher plants. Current Opinion in Plant Biology 2002, 5:164–172