Plastids
Plastids (derived from proplastids)
1.
2.
3.
4.
5.
6.
Chromoplast
Chloroplast
Amyloplast
Leucoplast
Elaioplast
Etioplast
In plants, meristamatic cells contain
10-14 proplastids, each carrying 12 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.
Plastid
nucleoid
ptDNA copies
The Structure of of O. sativa Chloroplast Genome
~120 genes
~50 transcription units
Two inverted repeats
One large single copy region
One small single copy region
These are salient
features of any higher
plant plastids
Conserved Features of Chloroplast Genomes in Higher Plants
Tobacco (155,939 bp)
LSC
IRB
SSC
IRA
86,686
25,341
18,571
25,341
Maize (140,387 bp)
82,355
22,748
12,536
22,748
Rice (134,525 bp)
80,592
Marchantia (121,024 bp)
81,095
Black pine (119,707 bp)
Epifagus (70,028)
20,799 12,334 20,799
65,696
19,799
10,058 19,813 10,058
495
53,021
495
22,735 4759 22,735
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
trn
Gene transcription
rpoA, B, C
ssb
Protein synthesis
rps2,3,4,7,8,11
rps12, 14, 15, 16, 18, 19
rpl2, 14, 16, 20, 22
infA
II. Photosynthesis
Photosynthetic proteins
rbcL
atpA, B, E
atpF, H, I
psaA, B, C
psbA, B, C, D, E
psbF, G, H, I
petA, B, D
Respiratory proteins
ndhA, B, C, D
ndhE, F
III. Others
Maturase
Protease
Envelope membrane protein
Ribosomal RNAs (16S, 23S, 4.5S, 5S)
Transfer RNAs (30 species)
RNA polymerase a, b, b’ subunits
ssDNA-binding protein
30S ribosomal proteins (CS) 2, 3, 4, 7, 8, 11
CS12, 14, 16, 18, 19
50S ribosomal proteins (CL) 2, 14, 16, 20, 22
Initiation factor I
RUBISCO large subunit
ATP synthetase CF1a, b, e subunits
ATP synthetase CF0I, III, IV subunits
Photosystem I A1, A2, 9-kDa protein
Photosystem II D1, 51 kDa, 44 kDa, D2, Cytb559-9kDa
Photosystem II Cytb559-4kDa, G, 10Pi, I proteins
Electron transport Cytf, Cytb6, IV subunits
NADH dehydrogenase (ND) subunits 1, 2, 3, 4
NDL4L, 5
matK
clpP
cemA
Organization of chloroplast genes into operons
Polycistronic mRNA
psbB
psbT
psbH
petB
Intron
psbN
Monocistronic
mRNA
petD
Intron
The Endosymbiont Theory
Common ancestor of plastid
and modern cyanobacteria
Flowering plant
Photosynthetic eukaryotic cell
Protoeukaryotic cell
Respiration
Common ancestor of mitochondira
and a-group of modern
proteobacteria
Photosynthetic
C-reduction
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
“-35”
Mustard
“-10”
psbA
TTGGTTGACATGGCTATATAAGTCATGTTATACTGTTCAAT
psbA
TTGGTTGACACGGGCATATAAGGCATGTTATACTGTTGAAT
rbcL
TGGGTTGCGCCATATATATGAAAGAGTATACAATAATGATG
atpB
TCTTGACAGTGGTATATGTTGTATATGTATATCCTAGATGT
trnM
TTATATTGCTTATATATAATATTTGATTTATAATCAATCTA
Spinach
Features of chloroplast transcription
1) Chloroplast promoters- similar to bacterial minimal promoter.
“-35”
Mustard
Spinach
“-10”
psbA
TTGGTTGACATGGCTATATAAGTCATGTTATACTGTTCAAT
psbA
TTGGTTGACACGGGCATATAAGGCATGTTATACTGTTGAAT
rbcL
TGGGTTGCGCCATATATATGAAAGAGTATACAATAATGATG
atpB
TCTTGACAGTGGTATATGTTGTATATGTATATCCTAGATGT
trnM
TTATATTGCTTATATATAATATTTGATTTATAATCAATCTA
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) Transcription
a) 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)
Endosymbiotic
Cyanobacterium
Photosynthetic
Cyanelle
Cyanophora paradoxa
Plastid transformation
Basic Requirements:
1)
Method of delivery (Biolistic method)
Micro-projectile
DNA-coated gold particles
Plate to stop
nylon projectile
Vents
Firing pin
Target cells
or tissues
Helium gas
2)
Nylon macro-projectile
Selectable marker (dominant marker)
Spectinomycin
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.
inhibits protein biosynthesis (70S ribosomes)
AMP
Spectinomycin
Adenylylspectinomycin (inactive protein synthesis inhibitor)
3”-adenylyltransferase
(aadA)
Construct:
“-35” “-10”
5’-UTR
SD
aadA-ORF
3’-UTR
A transformed plastid genome is formed by
two recombination events that are targeted by
homologous sequences. The plastid genome
segments that are included in the vector are
marked as the left (LTR) and right targeting
regions (RTR).
Chloroplast Genetic Engineering
Prokaryotic – No need for codon optimization
10, 000 copies per cell – high expression
Maternal inheritance
Not expressed in fruits ?
Multigene engineering
Homologous recombination
Multigene
Engineering
Maternal
Inheritance
Gene
Containment
Hyperexpression
Advantages of Chloroplast
Transformation
No Vector
Sequences
No Gene
Silencing
No Position
Effect
No
Pleiotropic
Effects
% Total Soluble Protein
A.
Cry2Aa2 Single Gene Expression
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Control
young
mature
old
B.
% Total Soluble Protein
Transgenic Leaf Age
Cry2Aa2 Operon Expression
50
40
30
20
10
0
Control young mature
old
Transgenic Leaf Age
100 fold higher expression obtained by plastid transformation (B)
compared to nuclear transformation (A)
Heteroplasmy vs homoplasmy
Accelerated gold particle
coated with transforming DNA
~10,000 plastid
genomes/cell
Several cycles
of
antibiotic
selection
(homoplasmy)
nucleus
biogenesis
sorting
Primary plastid
transformation
event (change
of
single plastid
DNA
molecule)
Cell and
organelle
divisions under
antibiotic
selection
(heteroplasmy)
chloroplast
proplastid
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
~60 copies of a single circular
chromosome per plastid
~50–60 chloroplasts per cell
Genes per chromosome
Two copies of each of
many chromosomes;
the number of
chromosomes per
diploid cell is species
-specific
Could be thousands
Arrangement and
transcription of genes
Each gene is separate
(individually transcribed )
Many genes are in operons
(transcribed together)
Chromosomes
~120–150
Selection markers
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.
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