Chloroplasts are double-membrane organelles found in plant cells that contain chlorophyll and are the site of photosynthesis. Chloroplast DNA is circular and ranges in size from 120,000 to 170,000 base pairs. It contains approximately 120 genes, including genes that encode proteins involved in photosynthesis and the transcription and translation machinery. Chloroplast DNA replication is semi-conservative and there are typically multiple copies of the chloroplast genome within each chloroplast.

1. Chloroplast genome organisation
BY,
S.NARTHANAA
192BO001
II MSC BOTANY
KASC

3. Chloroplast Definition
 “Chloroplast is an organelle that contains the photosynthetic pigment chlorophyll that captures sunlight and converts it into
useful energy, thereby, releasing oxygen from water. “
 Chloroplasts are found in all higher plants. It is oval or biconvex, found within the mesophyll of the plant cell. The size of
the chloroplast usually varies between 4-6 µm in diameter and 1-3 µm in thickness. They are double-membrane organelle
with the presence of outer, inner and intermembrane space. There are two distinct regions present inside a chloroplast known
as the grana and stroma.
 Grana are made up of stacks of disc-shaped structures known as thylakoids. The grana of the chloroplast consists of
chlorophyll pigments and are the functional units of chloroplasts.
 Stroma is the homogenous matrix which contains grana and is similar to the cytoplasm in cells in which all the organelles are
embedded. Stroma also contains various enzymes, DNA, ribosomes, and other substances. Stroma lamellae function by
connecting the stacks of thylakoid sacs.

4. Functions of Chloroplast
 The most important function of the chloroplast is to synthesize food by the process of photosynthesis.
 Absorbs light energy and converts it into chemical energy.
 Chloroplast has a structure called chlorophyll which functions by trapping the solar energy and used for the synthesis
of food in all green plants.
 Produces NADPH and molecular oxygen (O2) by photolysis of water.
 Produces ATP – Adenosine triphosphate by the process of photosynthesis.
 The carbon dioxide (CO2) obtained from the air is used to generate carbon and sugar during the Calvin Cycle or dark
reaction of photosynthesis.

5. Organization of Chloroplast
 Structural organization of chloroplast is signified by the presence of double membrane envelope and soluble phase, the
stroma, and an internal membrane system, the thylakoids. Both thylakoid and stromal systems are committed for light
reaction and carbon dioxide fixation respectively.
 Chloroplast attains diversified shapes. Higher plants exhibit lens shaped chloroplasts in their cytosol. The size
measures anywhere between 5 and 10 pm long.
 Stroma contains soluble enzymes known as rubisco (ribulose bisphosphate carboxylase-oxygenase), accountable for
upto 50% of the total leaf proteins. Its molecular weight of 500,000 consists of eight large subunits and eight small
subunits and it is credited with one of the most abundant available protein in nature. It executes photosynthesis by
accepting carbon dioxide as its substrate and reduces this to carbohydrate status.

6.  Several members of monocots show marginal deviation in their CO2 fixation process, generally known as C4 plants. The
maize, for example, is a C4 plant in which initial carbon dioxide fixation occurs in leaf mesophyll cells containing
chloroplasts, which lack rubisco and ultimately devoid of starch.
 The enzyme PEP carboxylase (phospho enol pyruvate carboxylase) acts as a major enzyme, catalyses first half of the
reaction by forming four carbon oxaloacetate, which is then converted into aspartic acid and malic acid which are exported
to bundle sheath cells, where they are decarboxylated and CO2 is refixed by bundle sheath due to the rubisco and operate
the Calvin cycle.
 In addition to their role in performing photosynthesis and carbon metabolism, chloroplasts are involved in other vital
functions such as the synthesis of amino acids and nucleotides, protein synthesis, pigments and hormones.

7. Chloroplast DNA
 Chloroplast DNA is comparatively large, circular in nature, commonly denoted
as ctDNA. The presence of DNA in chloroplast was first identified in 1962.
The size of chloroplast DNA is usually 140 kb in higher plants and less than
190 kb in lower eukaryotic plants. However, the size of the ctDNA is generally
between 120 and 155 kb.

8. History of cpDNA
 The presence of DNA in chloroplasts was first suggested during the early 1950s . Subsequent studies supported the
existence of extranuclear DNA in the chloroplasts of other plant species came about in the late 1950s and early 1960s.
In 1963, Masahiro R. Ishida, together with Ruth Sager, was acknowledged for being the first to extract the chloroplast
DNA. They were able to isolate chloroplasts from the alga, Chlamydomonas, and found an enriched satellite DNA that
has a buoyant density of 1,702 gm/cm3 and GC content of 39.3%.2 Soon, more DNA molecules were obtained from the
chloroplasts of higher plant species by other independent research teams.
 The demonstration of a unique DNA species in chloroplasts has led to intensive studies of both the structure of
chloroplast DNA and its expression. These studies have been accelerated by gene cloning and DNA sequencing
techniques developed in the mid-1970s.

9.  The first physical map of chloroplast DNA was constructed for maize in 1976 and the first chloroplast
gene was cloned in 1977. These studies and others established a new field, 'chloroplast molecular
biology,' and the organization and expression of chloroplast genomes were among the most extensively
studied fields in plant molecular biology.
 After 10 years the entire sequence of the chloroplast DNA was determined in tobacco, liverwort and
then in rice. Sequences for defined regions of many other chloroplast DNAs have also been completed,
but the identification and expression analysis of many chloroplast genes have mostly been done with
several representative higher plants and green algae.

12. Size of Chloroplast genomes
 Almost all chloroplast DNAs fall into the size range of 120 to 160 kb.
 Among chloroplast genomes for which an accurate size estimate exists, the siphonous green alga Codium fragile has the
smallest chloroplast DNA known (85 kb) while the green alga Chlamydomonas moewusii has the largest (292 kb).
 The chloroplast genome of the giant green alga Acetabularia is more complex than those of other plants and its genome
size appears to be 2000 kb.
 The population of chloroplast DNA in a plant species is generally homogeneous. However, the chloroplast genome of
the brown alga Pylaiella littoralis has been shown to be composed of two different circular DNA molecules of 133 kb
and 58 kb in size
 One of the outstanding features of the chloroplast DNAs found in most plants is the presence of a large inverted repeat
(IR) which ranges from 6 to 76 kb in length. Most of the size variation among land plant chloroplast DNAs can be
accounted for by changes in the length of the IR. The segments of the IR are separated by one large and one small
single-copy region (LSC and SSC, respectively).
 Pea, broad bean, alfalfa and pine chloroplast DNAs are exceptions to this pattern and lack IRs

13. Structure and Characteristics
 cpDNA is typically circular, and consists of base pairs ranging from 120,000 to 170,000 long. It has about 120
genes. Several copies of cpDNA molecules are present in each chloroplast. A chloroplast is one of the plastids, the
others are chromoplasts and leucoplasts. The chloroplasts are the photosynthetic type of plastid containing high
amounts of chlorophyll (the green pigment). The chloroplast has at least three membrane systems: outer membrane,
inner membrane, and thylakoid system (the site of photosynthesis). The stroma, which is the matrix of the
chloroplast, in between the grana contains cpDNA, enzymes, molecules, and ions. It is where the dark reactions of
photosynthesis occur. Most cpDNAs contain inverted repeats of about 4,000 to 25,000 base pairs long, with the
exceptions of pea plants and certain red algae that do not have inverted repeats in their cpDNAs.

14.  By employing DNA-binding flourescent dye several copies of the plastid genome have been visualized. The size of the chloroplast
genome can be comparable to bacteriophage T4 (165 kb). There are many copies of circular DNA in chloroplast, i.e., between 20
and 100 copies per chloroplast in higher plants.
 In higher plants, chloroplast DNA exists as double-stranded circular molecule. Unlike nuclear DNA, it does not contain 5-methyl
cytosine and is not associated with histones. Its buoyant density is around 1.690 gmL-1, which is corresponding to G + C ratio to
approximately 37 per cent.
 Measurement is based on DNA-DNA association through light on the potential coding capacity of the plastome. The molecular
weight of the plastid DNA is between 80 and 100 million, which corresponds between 12,000 and 150,000 base pairs

16.  Chloroplast contains one type of chromosome and assumes polyploid status. In young leaves, number of chloroplast attains 200 or
more. DNA replication in plastid is semi conservative. In chloroplasts of maize and pea, DNA replication begins at two sites about
7000 base pairs apart and proceeds in both the directions.
 Chloroplasts contain introns. They fall into two classes. One of the intron classes is located in tRNA genes and another class in
protein coding region. Several photosynthetic related genes that encode proteins are located in thylakoid membrane.
 Several evidences confirmed that chloroplast DNA contains 45 genes coding for RNA and 27 genes coding for proteins. These
proteins are mainly involved in chloroplast gene expression. The genes coding for proteins of the thylakoid membrane and another 10
gene products are committed for electron transport process.
 A restriction map for maize chloroplast DNA (139 kb) reveals that plastome contains unique 22,000 base pair inverted repeated
sequence, containing the rRNA genes (Fig.). Some other plastome with similar repeats contains two copies of rRNA genes.

18. The Characteristics of Chloroplast Gene
Expression
 The chloroplast gene-expression system is evolutionarily derived from photosynthetic bacteria that were endocytosed by
ancestral eukaryotic plant cells more than 1.5 billion years ago . During evolution, chloroplasts have retained core components
of the gene-expression apparatus from their prokaryotic progenitors. In addition, they obtained many eukaryotic properties, such
as RNA editing, the prevalence of introns, and complex processing patterns from polycistronic RNA precursors . Here, we
briefly describe the processes of chloroplast gene expression in plants .

20.  Overview of chloroplast gene expression. In plants, most chloroplast genes are organized as operons and
are controlled by single promoters (bent arrow). These genes are transcribed by two distinct types of RNA
polymerase: Nucleus-encoded RNA polymerase (NEP) and plastid-encoded RNA polymerase (PEP). The
resulting primary transcripts require several processing steps to form mature mRNA, including 50 and 30
trimming, intercistronic cleavage, RNA splicing, and RNA editing. In order for these events to take place,
numerous nucleus-encoded proteins are translated in the cytosol and imported into the chloroplast, where
they control and/or regulate chloroplast gene expression. Chloroplast gene translation is conducted by
bacterial-type 70S ribosomes, which occurs cotranscriptionally. Since the mRNA turnover rate within
chloroplasts is slow, most ribosomes function in posttranscriptional steps. Moreover, chloroplast gene
expression is involved in responses to environmental cues

21. Chloroplast ribosomes
 Chloroplast ribosomes contain about 50 ribosomal proteins, distributed between the two subunits. The 23 S, 5 S, 4.5 S
rRNA are present in the 50 S subunit and the 16 S rRNA is in the 30 S subunit. Plastid contains tRNA synthetase
enzymes. The presence of plastid tRNA is able to charge all of the 20 protein amino acids. Synthesis of protein in
chloroplast utilizes normal genetic code.
 The sequences of the maize and tobacco 16 S rRNA genes are 1491 and 1486 nucleotides in length, respectively. They
show 96% sequence homology with each other. Similarly, DNA sequence of 23 S rRNA genes from maize and tobacco
is 2898 and 2804 nucleotides respectively.
 The distance between 16 S (end) and the 23 S (start) of rRNA gene is 2408 base pairs in maize and 2080 in tobacco .
On the contrary, the distance among prokaryotic organisms is very less, for example, in E. coli distance is 440 base
pairs. Longer distance among higher plants is due to the presence of introns upto 950 base pairs.

22.  During transcription the 16 S, 23 S, 4.5 S rRNA sequence in chloroplast together with the tRNA in the spacer region
between 16 S and 23 S genes are transcribed as a polycistronic RNA, which is a precursor RNA undergoes modification to
produce mature tRNA and rRNA. Transcription of the rRNA genes takes place at promoter site by chloroplast RNA
polymerase upstream from the mature 16 S rRNA sequence and continues till end of the 4.5 S sequences.
 Post-transcriptional processing of rRNA such as intron splicing, generation of a number of RNA fragments, the ligation of
RNA sequence takes place. Information on the synthesis and processing of chloroplast mRNA is meagre. They seem to be
devoid of 5′ cap and do not contain long region of polyadenylic acid at the 3′ end. Some reports suggested that chloroplast
mRNA may contain short runs of oligo A

24. Plastid Regulatory Sequence
 Sequencing of plastid genes such as rbcL, rRNA, tRNA, CF polypeptides and photogene 32 have been
accomplished, of which rubisco large subunit gene from maize was the first to be sequenced (Mcintosh et al.,
1980). There are two putative promoter regulatory sequences (TTGATA and TATGA) present in this region.
 The putative regulatory sequence rbcL of other species shows deviation .

25. Expression of rbcL Gene in Chloroplast:
 Rubisco gene contains eight subunits of which four are smaller subunits and other four are larger subunits. The
genes for larger (L) sububits are coded in chloroplast DNA, and genes for smaller (S) subunits are coded in nuclear
DNA. In nuclear code genes have mRNA with 5′ cap and poly-A sequence as evidenced in rubisco gene.
 They are translated on cytosol ribosomes. The transit peptide, which varies from 40 to 60 amino acids in different
plants, is transported into chloroplast. After entry of eight smaller subunits inside the chloroplast, signal peptides
are cleaved and association between larger subunits and smaller subunits takes place to become functional
holoenzyme.

27.  There is a considerable imbalance between the number of nuclear-encoded genes for plastid function and number
of plastid-coded genes in photosynthetic cells of higher plants.
 Several hundreds of gene copies will be produced in chloroplast due to their high copy number; on the other
hand, nuclear DNA contains only few copies of the genes for photosynthetic functions.
 Inspite of this imbalance, some well coordination of gene expression could be seen in the chloroplast of higher
plants.

28. Biological function
 It is presumed that in due course some parts of the chloroplast genome were transferred to the nuclear
genome. The process is called endosymbiotic gene transfers. Because of this transfer, the chloroplast genome
is greatly reduced compared with that of cyanobacteria, which are conjectured as the ancestral origin of
chloroplasts.

33. Case studies
 Five Complete Chloroplast Genome Sequences from Diospyros: Genome Organization and
Comparative Analysis- journals.plos.org
Jianmin Fu et al[2016], https://doi.org/10.1371/journal.pone.0159566
Diospyros is the largest genus in Ebenaceae, comprising more than 500 species with remarkable economic value, especially Diospyros kaki Thunb., which has
traditionally been an important food resource in China, Korea, and Japan. Complete chloroplast (cp) genomes from D. kaki, D. lotus L., D. oleifera Cheng., D.
glaucifolia Metc., and Diospyros ‘Jinzaoshi’ were sequenced using Illumina sequencing technology. This is the first cp genome reported in Ebenaceae. The cp genome
sequences of Diospyros ranged from 157,300 to 157,784 bp in length, presenting a typical quadripartite structure with two inverted repeats each separated by one large
and one small single-copy region. For each cp genome, 134 genes were annotated, including 80 protein-coding, 31 tRNA, and 4 rRNA unique genes. In all, 179 repeats
and 283 single sequence repeats were identified. Four hypervariable regions, namely, intergenic region of trnQ_rps16, trnV_ndhC, and psbD_trnT, and intron of ndhA,
were identified in the Diospyros genomes. Phylogenetic analyses based on the whole cp genome, protein-coding, and intergenic and intron sequences indicated that D.
oleifera is closely related to D. kaki and could be used as a model plant for future research on D. kaki; to our knowledge, this is proposed for the first time. Further,
these analyses together with two large deletions (301 and 140 bp) in the cp genome of D. ‘Jinzaoshi’, support its placement as a new species in Diospyros. Both
maximum parsimony and likelihood analyses for 19 taxa indicated the basal position of Ericales in asterids and suggested that Ebenaceae is monophyletic in Ericales.

34. The complete nucleotide sequence of the
tobacco chloroplast genome: its gene
organization and expression-embopress.org
K. Shinozaki et al[1986] https://doi.org/10.1002/j.1460-2075.1986.tb04464.x
The complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It
contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single‐copy region. The genes
for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15
genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the
chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to
components of the respiratory‐chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes
are sufficient to read all codons if the ‘two out of three’ and ‘U:N wobble’ mechanisms operate in the chloroplast. Two sequences which
autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of
the chloroplast genes.

35. Conservation of chloroplast genome
structure among vascular plants-Springer
 Jeffrey D. Palmer & Diana B. Stein (1986)
 The first physical map of a gymnosperm chloroplast genome and compared its organization with those of a fern and several angiosperms by
heterologous filter hybridization. The chloroplast genome of the gymnosperm Ginkgo biloba consists of a 158 kb circular chromosome that
contains a ribosomal RNA-encoding inverted repeat approximately 17 kb in size. Gene mapping experiments demonstrate a remarkable similarity
in the linear order and absolute positions of the ribosomal RNA genes and of 17 protein genes in the cpDNAs of Ginkgo biloba, the fern Osmunda
cinnamomea and the angiosperm Spinacia oleracea. Moreover, filter hybridizations using as probes cloned fragments that cover the entirety of the
angiosperm chloroplast genome reveal a virtually colinear arrangement of homologous sequence elements in these genomes representing three
divisions of vascular plants that diverged some 200–400 million years ago. The only major difference in chloroplast genome structure among these
vascular plants involves the size of the rRNA-encoding inverted repeat, which is only 10 kb in Osmunda, 17 kb in Ginkgo, and about 25 kb in most
angiosperms. This size variation appears to be the result of spreading of the repeat through previously single copy sequences, or the reverse process
of shrinkage, unaccompanied by any overall change in genome complexity.

36. An update on chloroplast genomes-
Springer
 V. Ravi, J. P. Khurana, A. K. Tyagi & P. Khurana Published: 28 November 2007
Plant cells possess two more genomes besides the central nuclear genome: the mitochondrial genome and the chloroplast genome (or
plastome). Compared to the gigantic nuclear genome, these organelle genomes are tiny and are present in high copy number. These genomes
are less prone to recombination and, therefore, retain signatures of their age to a much better extent than their nuclear counterparts. Thus, they
are valuable phylogenetic tools, giving useful information about the relative age and relatedness of the organisms possessing them. Unlike
animal cells, mitochondrial genomes of plant cells are characterized by large size, extensive intra-molecular recombination and low
nucleotide substitution rates and are of limited phylogenetic utility. Chloroplast genomes, on the other hand, show resemblance to animal
mitochondrial genomes in terms of phylogenetic utility and are more relevant and useful in case of plants. Conservation in gene order, content
and lack of recombination make the plastome an attractive tool for plant phylogenetic studies. Their importance is reflected in the rapid
increase in the availability of complete chloroplast genomes in the public databases. This review aims to summarize the progress in
chloroplast genome research since its inception and tries to encompass all related aspects. Starting with a brief historical account, it gives a
detailed account of the current status of chloroplast genome sequencing and touches upon RNA editing, ycfs, molecular phylogeny, DNA
barcoding as well as gene transfer to the nucleus.

37. Methods for Obtaining and Analyzing
Whole Chloroplast Genome Sequences
(science direct)
 Robert K.Jansen et al.(2005)
During the past decade, there has been a rapid increase in our understanding of plastid genome organization and evolution due to the
availability of many new completely sequenced genomes. There are 45 complete genomes published and ongoing projects are likely to
increase this sampling to nearly 200 genomes during the next 5 years. Several groups of researchers including ours have been developing
new techniques for gathering and analyzing entire plastid genome sequences and details of these developments are summarized in this
chapter. The most important developments that enhance our ability to generate whole chloroplast genome sequences involve the generation
of pure fractions of chloroplast genomes by whole genome amplification using rolling circle amplification, cloning
genomes into Fosmid or bacterial artificial chromosome (BAC) vectors, and the development of an organellar annotation program (Dual
Organellar GenoMe Annotator [DOGMA]). In addition to providing details of these methods, we provide an overview of methods for
analyzing complete plastid genome sequences for repeats and gene content, as well as approaches for using gene order and sequence data
for phylogeny reconstruction. This explosive increase in the number of sequenced plastid genomes and improved computational tools will
provide many insights into the evolution of these genomes and much new data for assessing relationships at deep nodes in plants and other
photosynthetic organisms.