organisation and uses of chloroplast genome

Contents:
• What is chloroplast
• Ultrastructure of chloroplast
• Genome
• Physical properties
• Gene content
• Ct genes and encoded proteins
• Ultrastructure of ct genome
• Chloroplast transformation
• Recent uses of ct genome


Introduction-
Chloroplasts, as name suggests, are the plastids containing green pigment chlorophyll and is responsible for the green colour of photosynthetic organs of plants and algae. These are membrane bound, photosynthetic, eukaryotic organelle responsible for photosynthesis and observable as flat discs usually 2 to 10 µm in diameter and 1 µm thick. In land plants, they are, in general, 5 μm in diameter and 2.3 μm thick.

Ultrastructure of chloroplast-
The chloroplast is contained by an envelope that consists of an inner and an outer phospholipid membrane. Between these two layers is the intermembrane space. A typical parenchyma cell contains about 10 to 100 chloroplasts.

The material within the chloroplast is called the stroma, corresponding to the cytosol of the original bacterium, and contains one or more molecules of small circular DNA. It also contains ribosomes; however most of its proteins are encoded by genes contained in the host cell nucleus, with the protein products transported to the chloroplast Within the stroma are stacks of thylakoids, the sub-organelles, which are the site of photosynthesis. The thylakoids are arranged in stacks called grana (singular: granum). A thylakoid has a flattened disk shape. Inside it is an empty area called the thylakoid space or lumen. Photosynthesis takes place on the thylakoid membrane; as in mitochondrial oxidative phosphorylation, it involves the coupling of cross-membrane fluxes with biosynthesis via the dissipation of a proton electrochemical gradient.

1. outer membrane
2. intermembrane space
3. inner membrane (1+2+3: envelope)
4. stroma (aqueous fluid)
5. thylakoid lumen (inside of thylakoid)
6. thylakoid membrane
7. granum (stack of thylakoids)
8. thylakoid (lamella)
9. starch
10. ribosome
11. plastidial DNA
12. plastoglobule (drop of lipids

Chloroplast genome organization-
Chloroplasts posses a degree of autonomy within the cell that is in many ways similar to that of mitochondria. They do contain in the stroma a DNA that is unique to the organelle. With this genome a no. of chloroplast specific proteins are made using ribosomes that are also located in the stroma. Like mitochondria, chloroplasts replicate and thereby demonstrate a measure of reproductive autonomy.

Chloroplast contain their own genetic system, reflecting their evolutionary origins from photosynthetic bacteria. The 6 to 9 Mb genomes of present day free living photosynthetic cyanobacteria code for between 5400 and 7200 proteins. Like those of mitochondria, the genome of chloroplast consist of circular DNA molecules present in multiple copies per organelle. However, chloroplast genome are larger and more complex than those of mitochondria, ranging from 120 to 160 kb and containing approx. 150 genes.

Physical properties of chloroplast DNA-
• The entire chloroplast Genome resides within a single circular chloroplast DNA (ct DNA) molecule
• However, the DNA is generally present in multiple copies with as many as 20 to 60 ct DNA per chloroplast
• Depending on the species of organism, molecular weights of ct DNA commonly range from 85 to 140 ×106 daltons.
• The contour length is around 45µm, but may range from about 40 to 60 µm depending upon the species
• Isolated ct DNA exists in a variety of forms and conformations. Some are unicircular and other appeared as interlocked dimers.

• Two types of dimer are found:
-circular dimers which are formed by recombination between monomers, and
-catenated dimers in which two monomers interlink in a chain.
• Circular dimers may constitute upto 10% of ct DNA and catenated dimers about 2.5 %. The monomers often appear as relaxed circular duplexes in vitro, but in situ the closed supercoiled form is predominant.
• Ct DNA from higher plants and most algae have characteristic composition of 37+ 1 % (G+C).
• Density of 1.695 to 1.697 g cm-3. a notable exception is ct DNA from alga Euglena has G+C content of 28.2% and density of 1.685 g cm-3.


Gene Content of Chloroplast DNA-
The chloroplast genome of several plants have been completely sequenced, leading to the identification of many of the genes contained in the organelle DNAs.

First chloroplast genome sequenced was the liverwort (Marchantia polymorpha) genome which contains 121024 bp which contains 1024 inverted base repeats, separated by a small single copy region and a large single copy region.

These chloroplast genes code both RNAs and proteins involved in gene expression as well as variety of proteins that function in photosynthesis. Both the ribosomal and transfer RNAs used for translation of chloroplast mRNAs are encoded by the organelle genome


Genes encoded by the chloroplast DNA-
Function No. of genes

Genes for the genetic apparatus
rRNAs 4
tRNAs 30
Ribosomal proteins 21
RNA polymerase subunits 4

Genes for photosynthesis
photosystemI 5
photosystemII 12
Cytocrome bf complex 4
ATP synthase 6
Ribulose bis phosphate carboxylase 1

These include four rRNAs ( 23S, 16S, 5S and 4.5S). and 30 tRNA species. In contrast to the smaller no. of tRNAs encoded by mitochondrial genome, the chloroplast tRNAs are sufficient to translate all the mRNA codons according to the universal genetic code. In addition to these RNA components of the translation system, the chloroplast genome encodes about 20 ribosomal proteins, which represent approx. one third of the proteins of chloroplast ribosomes. Some subunits of RNA polymerase are also encoded by chloroplast, although additional RNA polymerase subunits and other factors needed for chloroplast gene expression are encoded in the nucleus.

The chloroplast genome also encodes approx. 30 proteins that are involved in photosynthesis, including the components of photosystem I and II, and of the cytochrome bf complex,, and of ATP synthase. In addition one of the subunits of ribulose bis phosphate carboxylase (rubisco) is encoded by chloroplast DNA. Rubisco is the critical enzyme that catalyzes the addition of CO2 to ribulose-1,5-bisphosphate during calvin cycle not only it is the major component of the chloroplast stroma, but it is also thought to be the single most abundant protein on earth, so it is noteworthy that one of its subunits is encode by the chloroplast genome.

Chloroplast Genes and protein encoded-
Ribosomal RNAs: Ribosomal RNA operons are designated as:
rrnA, B,C. Each operon normally includes genes for 16S rRNA, 23S rRNA, 5S rRNA, and 4.55 rRNA.

Transfer RNAs: tRNA genes are designated "trn' to indicate transfer RNA, followed by the single letter amino acid code indicating the amino acid accepted by the tRNA encoded by the gene. Where there is more than one gene for a particular amino acid, the isoaccepting species can be indicated either with sequential numbers or by giving the anticodon. About 40 tRNA genes are known to exist in the chloroplast genome.
Examples:

trnF-gene for tRNA(Phe)
trnC-gene for tRNA(Cys)
trn L1 (or trnL-UAA)--gene for tRNA(Leu)1
trn L2 (or trnL-CAA)--gene for tRNA(Leu)2

Ribosomal Proteins-

rps 4-ribosomal protein homologous to E. coli ribosomal protein S4.
rps 19---ribosomal protein homologous to E. coli ribosomal protein S 19
rpl 2-ribosomal protein homologous to E. coli ribosomal protein L2.

Photosystem I Proteins-
psaA 1-P700 chlorophyll a apoprotein
psaA2-P700 chlorophyll a apoprotein

Photosystem II Proteins-
psbA-"32 kilodalton' quinone-binding polypeptide. Also known as "photogene 32" and "Qb protein"; it contains the binding site for atrazine type herbicides.
psB-51 kilodalton chlorophyll a-binding polypeptide or p680 apoprotein.
pbC-44 kilodalton chlorophyll a-binding polypeptide.
pbD-"D2" protein.
psbE-cytochrome b559.

Photosynthetic Electron Proteins
pet-cytochrome f
petS-cytochrome b6
petD-subunit 4 of the cytochrome b6/f complex

Proteins of the ATP Synthase Complex-
atpA-CF1 alpha subunit
atpS-CF1 beta subunit
atpE-CF1 epsilon subunit
atpH-CFO subunit III, DCCD-binding proteolipid for the ATPase complex (proton translocating subunit)

Carbon Fixation Enzymes-
rbcL-ribulose bisphosphate carboxylase, large subunit
Other Stromal Polypeptides
tufA-translational elongation factor Tu

Ultra structure of chloroplast genome-
Chloroplast DNA contains long repetitive sequences making up 20 to 30 % of the contour length of the monomer. Shorter repetitive sequences that are inverted are also found in ct DNA.

The repetitive and non repetitive sequences are organized in segments in all ct DNA. The genome is thus divided into shorter regions, two of which contain repetitive sequences that are inverted with respect to each other and two that are made of nonrepetitive sequences . physical map of this sort have revealed that rRNA genes appear in the order 16, 23 and 5 S RNAs similar to that found in E. coli. Transcription is fron 16 to 23 S these two genomes are separated by approx. 2100 bp (in Zea mays). A 4.5S RNA, characteristic of chloroplast Ribosomes, is coded for by a gene in the vicinity of the 5S RNA genes.

Gene coding for chloroplast RNAs are scattered over the genome and are found both in inverted repeat regions and in non repetitive regions.

Chloroplast transformation (Transplastomic plant)-A transplastomic plant is a genetically modified plant in which the new genes have not been inserted in the nuclear DNA but in the DNA of the chloroplasts. The major advantage of this technology is that in many plant species plastid DNA is not transmitted through pollen, which prevents gene flow from the genetically modified plant to other plants.
Transformation technology-
The most common method to transform plastids is particle bombardment: Small gold or tungsten particles are coated with DNA and shot into young plant cells or plant embryos. Some genetic material will stay in the cells and transform them. The transformation efficiency is lower than in agrobacterial mediated transformation, which is also common in plant genetic engineering, but particle bombardment is especially suitable for plastid transformation.
In order to persist and be stably maintained in the cell, a plasmid DNA molecule must contain an origin of replication, which allows it to be replicated in the cell independently of the chromosome. Because transformation usually produces a mixture of rare transformed cells and abundant non-transformed cells, a method is needed to identify the cells that have acquired the plasmid. Plasmids used in transformation experiments will usually also contain a gene giving resistance to an antibiotic that the intended recipient strain of bacteria is sensitive to. Selection for cells able to grow on media containing this antibiotic can then select the cells that have acquired the plasmid by transformation, as cells lacking the plasmid will be unable to grow.

Transplastomic tobacco-However, plastid transformation is suitable only for certain crop species, and the reliability of this method has only been proven for tobacco. Led by Ralph Bock from the Max Planck Institute of Molecular Plant Physiology in Germany, researchers studied genetically modified tobacco in which the transgene was integrated in chloroplasts. The researchers analysed more than two million seedlings and found that less than 20 in 1,000,000 inherited the transgene. In the pollen of adult plants, the rate was even lower, remaining below 3 in 1,000,000. This reduction is because some parts of the seedlings are lost during their development into mature plants.
Because tobacco has a strong tendency towards self-fertilisation, the reliability of transplastomic plants is assumed to be even higher under field conditions. Therefore, the researchers believe that only one in 100,000,000 GM tobacco plants actually would transmit the transgene via pollen. Such values are more than satisfactory to ensure coexistence. However, for GM crops used in the production of pharmaceuticals, or in other cases in which absolutely no outcrossing is permitted, the researchers recommend the combination of chloroplast transformation with other biological containment methods, such as cytoplasmic male sterility or transgene mitigation strategies.

Recent applications of plastid genome-
• Expression of bar in the Plastid Genome Confers Herbicide Resistance-
Phosphinothricin (PPT) is the active component of a family of environmentally safe, nonselective herbicides. Resistance to PPT in transgenic crops has been reported by nuclear expression of a bar transgene encoding phosphinothricin acetyltransferase, a detoxifying enzyme. We report here expression of a bacterial bar gene (b-bar1) in tobacco (Nicotiana tabacum cv Petit Havana) plastids that confers field-level tolerance to Liberty, an herbicide containing PPT.

• Polymorphic simple sequence repeat regions in chloroplast genomes: applications to the population genetics of pines-
Analysis of 305 individuals from seven populations of Pinus leucodermis Ant. revealed the presence of four variants with intrapopulational diversities ranging from 0.000 to 0.629 and an average of 0.320. Restriction fragment length polymorphism analysis of cpDNA on the same populations previously failed to detect any variation. Population subdivision based on cpSSR was higher (Gst = 0.22, where Gst is coefficient of gene differentiation) than that revealed in a previous isozyme study (Gst = 0.05). We anticipate that SSR loci within the chloroplast genome should provide a highly informative assay for the analysis of the genetic structure of plant populations.

• Use as plastid vector-The present invention provides a method to circumvent the problem of using antibiotic resistant selection markers. In particular, the target plants are transformed using a plastid vectorwhich contains a heterologous DNA sequences coding for a phytotoxindetoxifying enzyme or protein. the selection process involves converting a antibiotic free phytotoxic agent by the expresed phytotoxin detoxifying enzyme or protein to yield a nontoxic compound. The invention provides various methods to use antibiotic free selection in chloroplast transformation.