In this article we will discuss about Chloroplast:- 1. Subject-Matter of Chloroplast 2. Origin of Chloroplasts 3. Structure 4. Organisation and Function 5. Chloroplast DNA 6. Protein Synthesis and Ribosomes.
Contents
Contents:
- Subject-Matter of Chloroplast
- Origin of Chloroplasts
- Structure of the Chloroplasts
- Organisation and Function of the Chloroplasts
- Chloroplast DNA
- Protein Synthesis and Ribosomes of Chloroplast
1. Subject-Matter of Chloroplast:
Living organisms depend on the degradation of carbohydrates and fats for getting their energy to perform metabolic activities. The general principle is by the oxidation of carbohydrates to carbon dioxide with the liberation of free energy.
Again the reduction of carbon dioxide is continually needed to maintain the earth’s food Supply. This has been accomplished through photosynthesis by green plants. Cells or plants that produce carbohydrate or reduce carbon through photosynthesis are known as Autotrophs and which cannot synthesise their food are known as Heterotrophs.
Although higher plants are photosynthetic organisms, the maximum amount of reduction of carbon is done by microorganisms or lower plants through photosynthesis. These organisms are blue-green algae and photosynthetic bacteria.
This metabolic process takes place by trapping the solar energy in the cytoplasmic organelles, such as chloroplasts in higher plants while the trapping of solar energy occurs within membranes in prokaryotes. But the mechanism of photosynthetic reactions is the same in both organisms.
2. Origin of Chloroplasts (Biogenesis):
Chloroplast also arises by division from the existing ones. This is very clear in algae, where one chloroplast divides into two during cell division. In higher plants, the division of chloroplasts is very difficult to observe as. the number of chloroplast is very high. Still, sometimes the dividing chloroplast is seen under the phase contrast microscope as in Spinach.
In case of meristematic cells, chloroplasts are not found. But these cells contain numerous small vesicles of about 1 µm in diameter which are known as Pro-plastids. These pro-plastids contain chloroplast DNA.
According to the nature of the cell, these pro-plastids differentiate into either amyloplasts (starch containing particles) or lipoplasts (filled with lipids) or proteinoplasts (filled with proteins). These are all colourless, hence they are also called Leukoplasts.
In presence of light, the pro-plastids differentiate into chloroplast (containing the green pigment chlorophyll):
In presence of light, pro-plastids are transformed into chloroplasts, with the enlargement and formation of tubules and invagination of the inner membranes. These tubules are finally formed as grana. But under etiolated conditions, invaginations of the inner membrane occur but they remain contracted into a compact structure known as Pro-lamellar body. This stage of pro-plastid is known as etioplast.
The conversion of these etioplasts to chloroplasts under light gives an excellent tool for the study of chloroplast biogenesis. The pigment present in the etioplasts differs from chlorophyll in having two hydrogen’s less in the porphyrin ring and, again, the phytol ring was not attached with the porphyrin ring.
This altered structure of pigment is known as Protochlorophyllide. In presence of light, phytol ring becomes attached with the porphyrin ring to produce chlorophyll as with the addition of two hydrogen atoms. But the bulk quantity of chlorophyll in the etioplasts is formed de novo after several hours of illumination.
The enzyme which is responsible for the de novo synthesis of the chloroplast is 5- aminolevulinate synthetase (ALA synthetase). Again, it has been noted that the induction of enzyme for the synthesis of chlorophyll occurs through activation of Phytochrome, the light- receptor pigment. This stimulation of enzymes requires a short exposure to red light.
Thus the chloroplast biogenesis occurs in different steps:
Step 1:
Breakdown of pro-lamellar body
Step 2:
Formation of thylakoids
Step 3:
Conversion of protochlorophyllide to chlorophyll
Step 4:
Induction of enzymes required for chlorophyll biosynthesis.
3. Structure of the Chloroplasts:
These cell organelles are found in green plants and have a definite role in photosynthesis. Chloroplasts are double-membrane with chlorophyll containing lamellar structure within the central soluble protein-rich components known as Stroma. Under the light microscope, chloroplasts show some small granules known as grana within a homogeneous stroma.
The diameter of each grana varies from 0.5-2.0 µm. The detailed structure of chloroplast has been understood with the help of electron microscope. The electron micrographs of chloroplast actually revealed the complex organisation of its membrane and internal structure. The membrane consists of two layers, outer and inner membrane with inter-membrane space.
The granular structure or the grana as seen through the light microscope shows the aggregation of flat membranous sacs or vesicles called Thylakoids. These sacs are found to be stacked one after another which are known as Stacked thylakoid or Grana Thylakoids. There are spaces within each sac or thylakoid which are referred to as Thylakoid spaces.
Some spherical particles are protruding from the thylakoid membranes showing similarity with those on the surface of the mitochondrial cristae. These spherical particles contain protein complex which has a significant role in the formation of ATP during photosynthesis. There are interconnections between stacked and un-stacked thylakoids, suggesting an entire internal membrane system of the thylakoids.
It has been found that thylakoid membranes are not continuous with the envelope of the chloroplast. But some interconnections are noted between the thylakoids and the inner membrane of the chloroplast through many small vesicles and tubules, known as Peripheral reticulum.
In Red algae, completely separate thylakoids, without any stacking, are noted in electron micrographs. But stacked thylakoids are found in higher green plants and green algae. In red and blue-green algae, granules containing pigments known as Phycobilisomes are present on the surface of the thylakoid membranes.
4. Organisation and Function of the Chloroplasts:
The organisation of chloroplasts is more or less similar to that of the mitochondria in many ways. Both these organelles are surrounded by a double-membrane with a complex system of internal membranes. Only difference in the membrane system is that thylakoids are not continuous with the inner membrane, while, in case of mitochondria cristae are continuous with the inner membrane of the envelope.
In both the organelles, spherical particles (F1S particles) are protruding from the inner membranes which help in the ATP synthesis. Cristae of the mitochondria do not form a separate compartment while the thylakoid space represents a distinct compartment.
The interrelationships between mitochondria, chloroplasts and peroxisomes are so similar in function that sometimes these three organelles remain closely associated with one another. Mitochondria sometimes provide ATP to chloroplasts particularly when plants are grown in very dim light.
Although, in the electron micrographs, the chloroplasts of higher plants show disc-shaped or elliptical structures (2-4µm in diameter and 10 µm in length) but they undergo morphological changes with regard to the physiological condition.
When chloroplasts are suddenly exposed to light from the dark conditions, they shrink in volume by about 50 per cent. Hence the structural alteration of the chloroplasts takes place in response to light.
Enzymes for carbon dioxide fixation and other dark reactions are present in the stroma and the enzymes for light reactions are present in the thylakoids. Two separate ways for carbon dioxide fixation are observed in higher plants which are broadly classified into C3 and C4 plants.
Plants of the C3 type (wheat, oats, soya bean, rice etc.) have chloroplasts with high starch content and with large number of grana. In C3 plants there is a special type of protein or enzyme known as RUBISCO (Rubulose 1, 5 bi-phosphate carboxylase- oxygenase) which helps in the addition of carbon dioxide to Ribulose bi-phosphate to form
3-phosphoglyceric acid (a 3-carbon compound) in the stroma. As 3-carbon compound is formed in this reaction it is called C3 pathway. This RUBISCO is a stromal protein with eight large sub-units of 13,000 molecular weight and eight small sub-units of 53,000 molecular weight. Large sub-units are generally active and small sub-units have a regulatory function.
In C4 plants (maize, sugarcane etc.) the carbon dioxide fixation occurs first in the mesophyll cells containing chloroplasts which do not store starch. It also lacks RUBISCO. The principle of reaction in these plants is to add 1 molecule of CO2 to one molecule of Phosphoenol pyruvate to form a four carbon compound Oxaloacetate with the help of an enzyme Phosphoenol pyruvate carboxylase (PEP carboxylase). As this pathway forms a 4-carbon compound, it is called C4 type.
Oxaloacetate is then converted to Malic acid and Aspartic acid. These are then decarboxylated after transportation to bundle sheath cells which contain chloroplasts having RUBISCO. The decarboxylated products like Pyruvate and Alanine go to mesophyll cells where they are again utilised in the C4 cycle.
The greater efficiency of C4 plants is possible due to the decarboxylation of Aspartic and Malic acid providing a high CO2 concentration in the bundle sheath cells. Thus the carboxylase activity in C4 plants is more than the oxygenase activity, i.e., the photorespiration rate is low.
(i) Light-Absorbing Pigments for Photosynthesis:
The process of photosynthesis starts with the absorption of solar energy by pigments present either in a specialised organelle, the chloroplast, or attached in the membrane. All photosynthetic cells or green cells contain chlorophyll which is responsible for the green colour of plants.
Light has properties of both waves and of particles. It is composed of discrete units of energy or quanta that are also called photons. The light energy is proportional to the frequency of the light. In biology, the light is referred to as wavelength of light (λ).
It has been noted that blue or red light (680 nm) is more photosynthetically active than green light. Chlorophyll a is the principal pigment involved in photosynthesis which is present in all higher plants and in green algae and blue- green algae (Table 3.14).
In higher plants and in green algae there is an additional chlorophyll molecule—chlorophyll b; chlorophyll c is present in brown algae, diatoms and chlorophyll d is present in red algae. Photosynthetic bacteria contain bacteriochlorophylls.
Chlorophyll a and chlorophyll b differ in their light-absorbing properties. The maximum absorption of light by chlorophyll a is at 420 and 663 nm while that of chlorophyll b is at 460 and 645 nm.
There are some additional photosynthetic pigments known as accessory pigments which also help in the absorption of light energy. These are Carotenoids and Phycobilins. Carotenoids absorb maximum light in the range of 400-500 nm wavelength of light (violet/blue-green region).
The colour of carotenoids in photosynthetic cells is masked by the green colour of the chlorophyll molecules but its colour is generally expressed in many vegetables like carrots and tomatoes.
Phycobilins are generally found in red and blue-green algae. The pigment present in red algae is known as Phycoerythrobilin while it is Phycocyanobilin in blue-green algae. These pigments remain in a complex form with some proteins to form Phycobilisome granules.
Accessory pigments cannot substitute the chlorophyll but it can extend the range of visible light to increase the photosynthetic efficiency. Emerson and Aronld showed in their experiments that about .0004 mol of CO2 is fixed per mole of chlorophyll molecule or, in other words, fixation of one molecule of CO2 requires about 2,500 molecules of chlorophyll.
When isolated chlorophyll in aqueous solution is irradiated with 650-700 nm of light, it fluoresces that means it releases photon of longer wavelengths. But when the chlorophyll is bound with the thylakoid membrane, it does not fluoresce but transfers energy (electron) to the neighbouring molecule.
This is one of the important processes of photosynthesis, i.e., the transfer of electron from the chlorophyll molecule to an electron acceptor which then acts as a reducing agent. The chlorophyll molecule then becomes a powerful oxidant having the property of removing electrons from other molecules to reform the original chlorophyll molecule.
In higher plants, four molecules of Oxidants (chlorophyll+) are capable of removing four electrons from (H20) to form (O2):
Blue-green algae can also oxidize water to form oxygen. But purple and green bacteria do not produce oxygen; instead they oxidize H2S to produce sulphur.
Here, excited chlorophyll removes electrons from H2S rather than water:
These bacteria can also use H2 gas as electron donor.
(ii) Photosystems in Chloroplasts to Absorb Light:
There are two photosystems in chloroplasts of higher plants to absorb photons. One is active at longer wavelengths (maximum absorption at 700 nm) of light known as Photosystem I (P700) while the other system is active at shorter wavelengths—known as Photosystem II (P680), i-e., maximum absorption of light occurs in 680 nm.
This differential response of the chlorophyll molecules is due to the difference in the formation of the chlorophyll-protein complex. Photosystem I is located in the thylakoid membranes while Photosystem II is present in the stacked regions of the thylakoid membrane, i.e., in the grana part of the chloroplast.
Each photosystem can be divided into two constituents:
(i) A core complex containing the reaction centre; and
(ii) A light harvesting complex (LHC).
The LHC is mostly associated with photosystem II and its primary function is to absorb light energy and also to transfer that energy to the reaction centres of the two photosystems. The mechanism of photosystems, i.e., electron-transfer chain, has been presented in a model which is referred to as the Z scheme.
All the intermediate electron carriers in this model are still unknown. Light is absorbed first by the Photosystem II which is then transferred to the reaction centre (Peso) of the same system. At the reaction centre, chlorophyll molecules become excited. Then the electrons from the excited chlorophyll molecule are transferred to a protein complex of Plastoquinone (Q). The photolysis of water takes place in this photosystem.
Then the electron from ‘Q’ is passed to the pool of plastoquinone molecules. Electrons are then transferred to Plastocyanin (PC) through cytochrome b and cytochrome (fc) and, finally, to Photosystem I. After absorption of light by Photosystem I, electrons are activated.
These are then passed to an Iron-Sulphur compound (FeS). From FeS, electrons are transferred to NADP through Ferredoxin with the help of the enzyme NADP reductase. NADP is thus reduced to NADPH. The electron gap or electron hole formed in this photosystem is filled up by the new electrons coming from Plastocyanin.
Hence, the flow of electrons goes on from water to NADP+ through two photosystems in presence of light. The absorption of light energy by these two photosystems helps to increase the redox potential from +0.82 V in water to -0.32 V (Oxidation-Reduction potential) in NADPH which liberates energy of about 50 K.cal. Some of this energy is also utilised in the synthesis of ATP.
(iii) Ultrastructure of Thylakoid Membranes:
Through Freeze-fracture microscopy, several particles are seen on the surface of the thylakoid membrane. In this freeze-fracture technique four different views of membranes can be seen: two surface views (outer and inner) are referred to as PS and PF, and two views of the fracture plane referred to as EF and ES.
Particles of 11 nm diameter are found on the outer surface (PS) of the thylakoid membrane. Two types of protein complex, referred to as CF1 and CF0, have been observed on the thylakoid membrane which show similarity with the F1 and F0 constituents of the mitochondrial ATP-synthesizing complex.
CFi is a hydrophilic protein while CF0 is a hydrophobic protein. CF1 is attached to the thylakoid membrane forming the spherical particles. It shows ATP synthetase activity, i.e., it helps in the synthesis of ATP. CF1 can be easily removed from the membrane. CF1 is anchored to the thylakoid membrane by the CF0 protein. The function of CF0 is to serve as a trans membrane channel through which protons can flow on their way to CF1 in initiating the synthesis of ATP.
The mechanism of the formation of electrochemical gradient or electron transfer chain is the same as in the mitochondria. Both the systems follow the chemiosmotic theory which states that the flow of electrons in the electron transfer chain drives the protons from one side of the membrane to the other.
In the thylakoid membrane there are four complexes involved in the transfer of energy or in the electron- transfer chain. These are arranged in the membrane in the following manner—Photosystem II, Cytochrome b-f complex, Photosystem I and, finally, CF1 – CF0 complex.
The whole system functions in such a way that two pairs of protons are freed into the thylakoid space each time a pair of electrons flow through the chain. Through freeze-fracturing techniques, it has also been found that PF face of the thylakoid membrane contains a group of small particles (8-11 nm in diameter).
The EF face contains a group of larger particles of 10-18 nm in diameter. These particles actually are the site of the characteristic protein complexes mentioned above of the thylakoid membranes. Of the four complexes, the first two, i.e., Photosystem I and Photosystem II, are responsible for light reactions.
The function of cytochrome b-f complex is in performing the electron-transfer between the two photosystems. The last one, i.e., CF1– CF0 complex, helps in the synthesis of ATP.
It has also been noted that stacked thylakoids have more photosystem II activity. This association of stacked thylakoids with photosystem II has a great functional significance because it involves with the light harvesting complex of photosystem II, i.e., in short, known as LHC II.
An important observation has been noted that the photosynthetic efficiency does not depend on the amount of chlorophyll present in the plastid but on the nature of thylakoid stacking. However, there is no proof that Porphyridium cruentum, a red alga without any thylakoid stacking, has lesser photosynthetic efficiency.
The smaller particles of the thylakoid membrane are the site of the PSI system. In this case, chlorophyll having light-absorbing capacity at 70 nm (P 700) is responsible for the conversion of light energy.
(iv) Photorespiration:
Photorespiration is a process associated with photosynthesis which uses up oxygen and liberates CO2 in presence of light. It reduces the efficiency of photosynthesis to some extent as this process oxidizes the carbon compound produced during photosynthesis.
Hence the photorespiration can be referred to as a wasteful process for the economy of the plant. The process involves the oxidation of 1,6 Ribulose bi-phosphate (RUBP) by molecular oxygen, when oxygen concentration is high, RUBP is oxidised to phosphoglycolate and 3-Phosphoglycerate.
Phosphoglycolate is transformed into Glycolate which is transferred to Peroxisomes (Glyoxysomes)—smaller organelles containing a number of enzymes that generate and consume H2O2—for further oxidation. Glycolate in the Peroxisomes undergoes several reactions to finally transform into-an amino acid, glycine.
This glycine is then transported to mitochondria where it breaks down into C02 and the amino acid Serine. Serine comes back to Peroxisomes, where it converts into Glycerate. Glycerate then is returned to the chloroplast to enter the Calvin cycle. Photorespiration requires the involvement of three organelles like chloroplasts, mitochondria and peroxisomes and it is a cyclic process (Fig. 3.15).
Again, photorespiration is absent in C4 plants and so the photosynthetic efficiency of these plants is high. The absence of photorespiration in C4 plants is due to the high concentration of CO2 delivered to the Calvin cycle which prevents O2 to come into the process of photorespiration.
But the rate of photorespiration in C3 plants is high causing low photosynthetic efficiency. The actual reason for this is not clear. One possibility is that it is to reduce the toxic effect of high concentration of oxygen.
(v) Photosynthetic Process in Prokaryotes:
The photosynthetic process of blue-green algae is more or less similar to that of higher plants. But this process is different in purple and green bacteria. In case of blue-green algae flattened membranous sacs are present like the thylakoid membranes of higher plants.
The association of pigments like Phycobilin in the form of Phycobili some granules is found with the thylakoid membranes. The pigment Phycobilin absorbs most of the light energy which is then passed to the chlorophyll molecules located within the thylakoid membranes.
The photosynthetic process, i.e., light and dark reactions, are similar to that of higher plants. The light reactions in the purple and green bacteria occur in the photosynthetic membranes, known as Photosynthetic lamellae or Chromatophores within the cytoplasm.
These structures are formed through the invagination of the plasma membrane, forming stacks of membranes like grana. Sometimes the green bacteria form spherical vesicles instead of stacking of membranes.
Salient Features of Bacterial Photosynthesis:
1. Bacteria generally utilise light of longer wavelength (840-1,000 nm).
2. Presence of a single photosystem.
3. NAD+ used as electron acceptor instead of usual NADP+.
4. Electron donor is not water, the most common electron donor is H2S (Hydrogen Sulfide).
5. Oxygen is produced by the light reactions as water molecules are not used.
6. In the dark reactions also, the electron acceptor is different.
7. Electrons derived from the light reactions of photosynthesis are utilised to reduce hydrogen ions to H2 and atmospheric Nitrogen to Ammonia.
Another bacteria, Halo bacterium holobium, have a light-sensitive protein called Bacteriorhodopsin to utilise solar energy in the formation of ATP. This pigment containing protein is present on the surface of the membrane forming large purple patches.
The understanding of all these natural photosynthetic processes through trapping of solar energy may help in preparing one artificial light-trapping system in future that may substitute the conventional energy source.
5. Chloroplast DNA:
Ris and Plaut first demonstrated DNA in the chloroplast in 1962. Electron microscopic photograph showed its fibrillar nature. This DNA can be seen under light microscope in certain cases using DNA-binding fluorescent dyes. DNA form nucleoids within the stroma though it remains attached with the chloroplast membrane.
Chloroplast DNA (Cp DNA) is a double- stranded circular in nature without any association with histones. 5-methyl cysosine is absent in Cp DNA. The percentage of G-C in the chloroplast DNA varies from 36-40% in different species.
The re-association rate of chloroplast DNA with nuclear DNA varies from 10 to 20%. The size of the chloroplast DNA is between 120,000 and 150,000 base pairs with molecular weight between 80 and 100 million. There are many copies of chloroplast DNA which is 200 copies or more.
The replication of Cp DNA takes place in a semi-conservative way. In case of Maize and Pea, the replication starts’ at two loci having distance between them as 7,000 base pairs. The autonomous replication sequences like ars sequences of yeast has been reported in the chloroplast DNA of tobacco.
The complete DNA sequence of chloroplast DNA has been determined in case of Marchantia polymorpha and tobacco (Ohyama and others, 1986; Shinozaki, 1986). With the help of the hybridisation experiments of DNA-RNA, many protein coding genes, positions of many tRNAs and rRNAs of the chloroplast ribosomes have been identified.
The size of the chloroplast DNA of tobacco is of 155, 844 base pairs and have the code for about 146 genes. Chloroplast DNA of Marchantia has 128 genes with 121,024 base pairs. Chloroplast DNA contains a pair of inverted repeats which encodes generally the rRNA genes.
There are some ORFs (open reading frames) with coding sequences beginning with a met-codon and a stop codon at the end in the chloroplast DNA. In tobacco, genes for 23S, 16S, 4.5S and 5S ribosomal RNA and about 30 tRNAs have been detected.
Codes for some ribosomal proteins (rpl and rps), some sub-units of RNA polymerase (rpo A, B, C) and some large sub-units of RUBISCO (rbc L) have been identified in chloroplast DNA of tobacco. Two genes for the polypeptides of PSI and eight for the polypeptides of PSII have been identified.
Some herbicide-resistant genes have also been located in the Cp DNA. It has also been noted that some chloroplast DNA contains some genes which show homology with NADH dehydrogenase from human mitochondria.
6. Protein Synthesis and Ribosomes of Chloroplast:
Lyttleton first showed that chloroplasts contain 70S ribosomes whereas 80S ribosomes are present in the cytoplasm. Chloroplast ribosomes show more similarity with bacteria and blue-green algae. Ribosomal proteins of the chloroplast are encoded both by the nucleus and the chloroplast DNA showing their interdependence. The structure of the chloroplast tRNAs show more similarity with that of prokaryotic cells.
Chloroplasts have about 30 tRNAs genes which help in translating chloroplast-encoded RNA. The genetic code used by Cp DNA is the same as that of nuclear DNA. With the help of two protein inhibitors, cycloheximide and chloramphenicol, it has been noted that the synthesis of large sub-unit is only inhibited by chloramphenicol and the synthesis of small sub-unit is inhibited by cycloheximide.
This shows that small sub-unit is controlled by the nuclear gene and the large sub-unit is controlled by the chloroplast gene. The large sub-unit of RUBISCO was the first polypeptide synthesised among the chloroplast proteins. Besides this, about 60 polypeptides have been synthesised by chloroplasts. It has also been confirmed that small sub-unit of RUBISCO is not synthesised by isolated chloroplasts.
The composition and regulation of chloroplast RNA polymerases are not fully known. These are found to be coded by nuclear genes. But 3 genes have been identified (α β and β sub-units) in tobacco chloroplast DNA which shows similarity with the sub-units of E. coli RNA polymerase.
The molecular weights of these sub-units are 39,000, 121,000 and 155,000 respectively. One transcription factor—known as S factor, has been identified in maize chloroplast which helps to transcribe certain genes preferentially and also the large sub-unit RUBISCO gene.
The promoter sequences for transcription initiation has been found in E. coli genes at a distance of 10 and 35 base pairs upstream from the transcript. Similar sequences have been observed in chloroplast genes. In case of Spinach chloroplast, two promoter sequences for RNA polymerase have been found.
These are Cpt 1 and Cpt 2 having sequences as TTGCTT and TATAAT, respectively. Cpt 1 and Cpt 2 sequences are 77 and 53 base pairs upstream from the transcription starting point.
Transport of Some Proteins Into Chloroplasts:
It has already been established that some nuclear genes are involved in the function of the chloroplast. Some of these nuclear genes are the small sub-unit of RUBISCO (rbc S), ferredoxin, ferredoxin NADP oxidoreductase, plastocyanin, Cab polypeptides, photosynthetic reaction centres and ATPase etc. Hence there is a mechanism by which proteins synthesised in the cytoplasm are transported into the chloroplast.
The translation of nuclear encoded mRNAs are made on 80S ribosome of the cytosol. After the transcription, additional amino acids are attached to the N terminus of the mature protein. They also contain information for recognition and transport of the proteins by the chloroplast membranes.
The transport of small sub-units from the cytoplasm and the formation of RUBISCO assembly in the chloroplast with the combination of large sub-unit has been diagrammatically shown (Fig. 3.16).
