Plastids
Plastids (derived from proplastids)
1.
Chromoplast
2.
Chloroplast
3.
Amyloplast
4.
Leucoplast
5.
Elaioplast
6.
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.
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
LSC
IR
B
SSC
IR
A
86,686
25,341
18,571
25,341
82,355
22,748
22,748
12,536
80,592
20,799
20,799
12,334
81,095
10,058
10,058
19,813
65,696
495
495
53,021
19,799
22,735
22,735
4759
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 transcription
rpoA, B, C
RNA polymerase
a
,
b
,
b
’ subunits
ssb
ssDNA
-
binding protein
Protein synthesis
rps2,3,4,7,8,11
30S ribosomal proteins (CS) 2, 3, 4, 7, 8, 11
rps12, 14, 15, 16, 18, 19
CS12, 14, 16, 18, 19
rpl2, 14, 16, 20, 22
50S ribosomal proteins (CL) 2, 14, 16, 20, 22
infA
Initiation factor I
II. Photosynthesis
Photosynthetic proteins
rbcL
RUBISCO large subunit
atpA, B, E
ATP synthetase CF1
a
,
b
,
e
subunits
atpF, H, I
ATP synthetase CF
0
I, III, IV subunits
psaA, B, C
Photosystem I A1, A2, 9
-
kDa protein
psbA, B, C, D, E
Photosystem II D1, 51 kDa, 44 kDa, D2, Cytb559
-
9kDa
psbF, G, H, I
Photosystem II Cytb559
-
4kDa, G, 10Pi, I proteins
petA, B, D
Electron transport Cytf, Cytb6, IV subunits
Respiratory proteins
ndhA, B, C, D
NADH dehydrogenase (ND) subunits 1, 2, 3, 4
ndhE, F
NDL4L, 5
III. Others
Maturase
matK
Protease
clpP
Envelope 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 plastid
and modern cyanobacteria
Common ancestor of mitochondira
and
a
-
group of modern
proteobacteria
Protoeukaryotic cell
Photosynthetic eukaryotic cell
Flowering plant
Photosynthetic
C
-
reduction
Respiration
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 TTGGTTGACATGGC
TATATAA
GTCATGTTATACTGTTCAAT
psbA TTGGTTGACACGGGCA
TATAA
GGCATGTTATACTGTTGAAT
rbcL TGGGTTGCGCCA
TATATA
TGAAAGAGTATACAATAATGATG
atpB TCTTGACAGTGG
TATAT
GTTGTATATGTATATCCTAGATGT
trnM TTATATTGCTTA
TATATAA
TATTTGATTTATAATCAATCTA
Mustard
Spinach
“
-
35”
“
-
10”
1) Chloroplast promoters
-
similar to bacterial minimal promoter.
psbA TTGGTTGACATGGC
TATATAA
GTCATGTTATACTGTTCAAT
psbA TTGGTTGACACGGGCA
TATAA
GGCATGTTATACTGTTGAAT
rbcL TGGGTTGCGCCA
TATATA
TGAAAGAGTATACAATAATGATG
atpB TCTTGACAGTGG
TATAT
GTTGTATATGTATATCCTAGATGT
trnM TTATATTGCTTA
TATATAA
TATTTGATTTATAATCAATCTA
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) 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)
2)
Selectable marker (dominant marker)
Firing pin
Helium gas
Nylon macro
-
projectile
Micro
-
projectile
DNA
-
coated gold particles
Vents
Plate to stop
nylon projectile
Target cells
or 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 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
Advantages of Chloroplast
Transformation
Gene
Containment
Maternal
Inheritance
No Gene
Silencing
No Position
Effect
Hyper
-
expression
Multigene
Engineering
No Vector
Sequences
No
Pleiotropic
Effects
Cry2Aa2 Single Gene Expression
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
Control
young
mature
old
Transgenic Leaf Age
% Total Soluble Protein
Cry2Aa2 Operon Expression
0
10
20
30
40
50
Control
young
mature
old
Transgenic Leaf Age
% Total Soluble Protein
A.
B.
100 fold higher expression obtained by plastid transformation (B)
compared to nuclear transformation (A)
Accelerated gold particle
coated with transforming DNA
~10,000 plastid
genomes/cell
Primary plastid
transformation
event (change
of
single plastid
DNA
molecule)
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 circular
many chromosomes;
chromosome per plastid
the number of
~50
–
60 chloroplasts per cell
chromosomes 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
Enter the password to open this PDF file:
File name:
-
File size:
-
Title:
-
Author:
-
Subject:
-
Keywords:
-
Creation Date:
-
Modification Date:
-
Creator:
-
PDF Producer:
-
PDF Version:
-
Page Count:
-
Preparing document for printing…
0%
Σχόλια 0
Συνδεθείτε για να κοινοποιήσετε σχόλιο