chloroplast being the second semi-autonomous organelle of the plant cell also harbours its genome. the presentation includes various characteristic features of this organelle genome along with its functional pecularities and significance

1. DR. VIBHA KHANNA
ASSO. PROF. (BOTANY)
S.P.C. GOVERNMENT COLLEGE
AJMER (RAJASTHAN)

2. CYTOGENETICS
• BLOCK 1
• PRESENTATION 3:
CHLOROPLAST GENETICS

3. ORIGIN OF PLASTIDS
• The first plastid is thought to have originated through
primary endosymbiosis, in which a photosynthetic
cyanobacterium was captured by a heterotrophic protist
and eventually transformed into an intracellular organelle.
• Molecular clock analysis suggests this key event in
eukaryote evolution occurred approximately 1.7 billion
years ago (bya); early Devonian age.
• Cyanobacteria originated around 3.5bya.
• Plastids are considered as symbiont of past Cyanobacteria,
where most of its DNA has been transferred into the
nuclear genome and only 120-140 genes have been left out
in chloroplast genome.
https://www.slideshare.net/vibhakhanna1/endosymbiotic-
theory

4. Plastids: the semi-autonomous organelles
• Like mitochondria, even plastids evolved from bacteria
that were engulfed by nucleated ancestral cells.
• As a relic of this evolutionary past, both types of
organelles contain their own genomes, as well as their
own biosynthetic machinery for making RNA and
organelle proteins.
• Mitochondria and plastids are never made de novo, but
instead arise by the growth and division of an existing
mitochondrion or plastid.
• Mitochondrial and plastid proteins are encoded in two
places: the nuclear genome and the separate genomes
harbored in the organelles themselves.

5. The similarities between the genomes of
chloroplasts and bacteria:
• Chloroplast ribosomes are very similar to E. coli ribosomes,
both in their structure and in their sensitivity to various
antibiotics (such as chloramphenicol, streptomycin,
erythromycin, and tetracycline).
• In addition, protein synthesis in chloroplasts starts with N-
formyl methionine, as in bacteria, and not with the methionine
used for this purpose in the cytosol of eucaryotic cells.
• The basic regulatory sequences, such as transcription
promoters and terminators, are virtually identical in the two
cases.
• The amino acid sequences of the proteins encoded in
chloroplasts are clearly recognizable as bacterial and several
clusters of genes with related functions (such as those
encoding ribosomal proteins) are organized in the same way in
the genomes of chloroplasts, E. coli, and cyanobacteria.

6. Promiscuous DNA:
• Many of the genes of the
original bacterium are now
present in the nuclear
genome, where they have
been integrated and are
stably maintained. In higher
plants, for example, two-
thirds of the 60 or so
chloroplast ribosomal
proteins are encoded in the
cell nucleus; these genes
have a clear bacterial
ancestry, and the
chloroplast ribosomes
retain their original
bacterial properties.

7. Characteristics of Chloroplast DNA
{cp/ct DNA}
• Most not all plastids have double stranded, circular DNAs of
120-140kbps.
• Some show linear forms example in Maize 10-14 days old
plastid DNA is linear and exhibit the ends of linear genomic
monomers and head-to-tail (h–t) concatemers* within
inverted repeat sequences (IRs) near probable origins of
replication.
• Chloroplast DNA exists in the form of protein associated
loops called Nucleoids.
• Chloroplast DNA is compacted with histone like proteins.
• Nucleoids of Chloroplast are enriched with proteins
involved in DNA replication, organization and repair as well
as transcription, mRNA processing, splicing and editing.

8. Characteristics of Chloroplast DNA
• The size of the genome has been
determined for a number of plants
and algae and ranges from 85 to
292 kilobase pairs, with most being
between 120 kb and 160 kb.
• The chloroplast
genome organization is very similar
in all higher plants, although the
size varies from species to
species—depending on how much
of the DNA, surrounding the genes
encoding the chloroplast's 16S and
23S ribosomal RNAs, is present in
two copies.

9. Characteristics of Chloroplast Genome:
• cp/ct genome varies widely in copy number, from a few copies in seeds
and root cells, to very high copy number in young, rapidly growing leaf
cells.
• As the leaves mature the genome copy number drops, suggesting a
mechanism for control of cp (ct) DNA replication, but not linked to the
nuclear genome replication or cell cycle.
• These organelles contain a genome that encodes several proteins for
chloroplast function, but majority of the essential proteins are encoded
by the nuclear genome and are imported into the chloroplast.
• The mRNA transcripts of the chloroplast genes are translated according
to the standard genetic code.
• All components of the chloroplast DNA (ctDNA) replication machinery
appear to be nuclear encoded, including the DNA polymerase(s) and
accessory proteins such as DNA primase, DNA helicase, SSBs,
topoisomerases, and other factors.
• Mitochondria import most of their lipids; chloroplasts make most of
theirs

10. CHLOROPLAST DNA

11. The Chloroplast DNA:
• Structurally the chloroplast DNA consists of two IR
sequences, flanking the large and small single copy
sequences.
• It is of, more or less, constant size ranging from 120 to
160kb and contain nearly 120 genes.
• The chloroplast genes code for various RNAs and
proteins involved in transcription and translation of its
genome.
– It encodes all the ribosomal and transfer RNAs i.e. the four
rRNAs: 23S, 16S, 5S and 4.5S and
– the 30 tRNAs, of the chloroplast.
• Along with this the chloroplast genome also code for
nearly one third of the proteins of chloroplast
ribosomes( approximately 20 ribosomal proteins).

12. The Chloroplast DNA:
• Some of the subunits of RNA polymerase are
also transcribed and translated within the chloroplast the
remaining being transcribed by the nuclear genome.
• Approximately 30 different proteins involved in
photosynthesis are encoded by the chloroplast genome.
They include components of photosystem I and II, of the
cytochrome bf complex and the ATPase.
• The enzyme ribulose biphosphate carboxylase (RuBP
carboxylase), i.e. RUBISCO, the most abundant protein on
earth, is composed of two polypeptides (subunits): The
larger polypeptide, called rbcL, is a product of a chloroplast
gene, whereas the smaller polypeptide is the product of a
nuclear gene.

13. The Chloroplast DNA:
• Similarly enzyme ATPase, [the enzyme that uses proton
gradient energy to produce the important energy
molecule adenosine triphosphate (ATP)], comprises nine
different polypeptides.
Six of these polypeptides are products of chloroplast genes,
but the other three are products of nuclear genes that
must be transported into the chloroplast to join with the
other six polypeptides to make active ATPase.
• Nearly, thirty or so genes remain unidentified. Their
presence is inferred because they have DNA sequences that
contain all the components found in active genes. These
kinds of genes are often called “open reading frames”
(ORFs) until the functions of their polypeptide products are
identified.

14. Significance of the chloroplast genome:
• Phylogenetic analysis: Of greater importance has been the discovery that
the DNA sequences of many chloroplast genes are highly conserved; i.e.,
they have changed very little during their evolutionary history.
This fact has led to the use of chloroplast gene DNA sequences for
reconstructing the evolutionary history of various groups of plants.
{One of the most widely used sequences is the rbcL gene. It is one of the
most conserved genes in the chloroplast genome}
• Breeding: Variation in the sequence of chloroplast genome has been
utilised for understanding the origin, geographic distribution and climatic
adaptations of economically important crops. This has been made use of
in plant breeding experiments and various conservation strategies
developed for them.
• An important application of the chloroplast genome in agriculture is the
determination of purity of various commercial cultivars and identification
of closely related cultivars which are genetically compatible.
• Genetic engineering: Chloroplast genome has been used for the
development of highly efficient transformation vectors for genetic
engineering.

15. Optimizing foreign gene expression in
chloroplast genome:
• Chloroplast genome has been used for the
development of highly efficient transformation vectors
for the integration and expression of foreign genes.
This is achieved by integrating the gene of interest in
the intron regions which are flanked by the chloroplast
genes. This facilitates the integration and expression of
the transgenic cassettes. The cassette includes the
marker and the regulatory sequences required for the
expression.
• It minimizes out-crossing of transgenes to related
weeds or crops and also reduces the potential toxicity
of transgenic pollen to non-target insects.

16. Chloroplast Genome Engineering:
• Inheritance of the chloroplast genome is not in accordance with the
Mendelian principles and hence engineering the chloroplast genome is a
convenient approach. Successful stories of the chloroplast genome
engineering includes:
– Conferring resistance against biotic and abiotic stress i.e., resistance to
herbicides, insects, disease and drought:
• Trehalose is a non-reducing disaccharide of glucose which accumulates under stress
conditions such as freezing, heat, salt or drought and protects against damage imposed
by these stresses. The yeast trehalose phosphate synthase (TPS1) gene has been
successfully introduced into the tobacco chloroplast . This compartmentalization of
trehalose within chloroplasts confers drought tolerance without undesirable phenotypes.
– Development of edible vaccines and biopharmaceuticals:
• The ability to express foreign proteins at high levels in chloroplasts and chromoplasts,
and to engineer foreign genes without the use of antibiotic resistant genes, make the
chloroplast ideal for the development of edible vaccines and biopharmaceuticals.
• Chloroplasts, with their highly polyploid genomes offer an ideal compartment for
overproduction of the foreign proteins. An additional advantage of using chloroplasts is
their ability to process eukaryotic proteins, including folding and formation of disulfide
bridges.
• This has led to development of oral delivery of polio vaccine and proinsulin. It has also
enchanced the nutritive value of various edible products.
– Successful stories of chloroplast genome engineering also includes
accumulation of PHB ( Polyhydroxy Butyrate: a biodegradable polymer) in the
leaves of various plants.

17. GLOSSARY
• An inverted repeat (or IR) is a single stranded
sequence of nucleotides which is followed
downstream by its reverse complement.
• Concatemer is a long continuous DNA
moleculethat contains multiple copies of the
same DNA sequence linked in series)
• One kilobase (kb) equals one thousand base
pairs.

18. ?????!....Promiscuous DNA:
• The discovery that certain key chloroplast proteins, such as
ATPase and RuBP carboxylase, are composed of
a combination of polypeptides coded by chloroplast and
nuclear genes also raises some as yet unanswered
For example, why would an important plant structure like
the chloroplast have only part of the genes it needs to
function?
Moreover, if chloroplasts, as evolutionary theory suggests,
were once free-living bacteria-like cells, which must have
had all the genes needed for photosynthesis, why and how
did they transfer some of their genes into the nuclei of the
cells in which they are now found?

19. REFERENCES
• Alberts B, Johnson A, Lewis J, et al. Molecular
Biology of the Cell. 4th edition. New York:
Garland Science; 2002. The Genetic Systems
of Mitochondria and Plastids. Available from:
https://www.ncbi.nlm.nih.gov/books/NBK269
24/
• T.A. Brown. Genetics: A Molecular Approach.