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The most important function of chloroplast is photosynthesis which is essentially divided into two reactions: Light reaction which takes place in the thylakoid ...
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The term plastids is derived from Greek word “plastikas” meaning formed or moulded. The term was first used by Schimper in 1885. Based on the presence and absence of pigments he classified plastids into two categories:
1. Leucoplasts (l euco =white): the plastids lack pigments are known as leucoplasts. They are mainly storage plastids and are further classified based on the nature of stored product.
a) Amyloplasts: Store starch b) Aleuroplasts/proteinoplast: store protein c) Elaioplasts/Oleosomes: store fats
2. Chromoplast ( chroma =color): These are colored plastids. They can be further classified based on the type of pigment.
a) Chloroplast: these are green colored plastids and contain chlorophyll pigment. b) Rhodoplast: they contain pigments like fucoxanthin and are found in brown algae. c) Phaeoplasts: they contain pigments like phycoerythrin and are found in red algae.
Schimper suggested that different types of plastids can be interconverted e.g. chloroplast can be converted to chromoplast during fruit ripening. All plastids arise from undifferentiated organelles known as proplastids which are 0.5-1 μm in diameter. Depending on the cell’s requirement, proplastids can be converted into various types of plastids. The development of the plastids depends on various external and internal signals. Chloroplasts develop only in the presence of light from the vesicles budding from the inner chloroplast membrane. An intermediate form known as etioplast is seen when the plants are kept in dark which is characterized by the array of membrane tubules that lack chlorophyll.
Chloroplasts are found in green plant parts like leaves and stems. These are absent from animals, fungi and bacteria.
The size of chloroplast varies from 4 to 10 μm. The size may be controlled by genetic and several other factors. Polyploid cells have usually larger sized chloroplasts as compared to diploid cells, same is the case with the plants grown in shades which have larger sized chloroplast as compared to those grown in sunlight.
The stroma also contains third membrane system forming flattened discs stacked known as thylakoids. The thylakoids are stacked to form grana. The adjacent grana are connected by means of stromal lamellae or frets. Thylakoid membranes are rich in protein but contain relatively small amounts of phospholipid. Thylakoid membranes contain considerable amount of glycolipids and double bonds making it highly fluidic. Also thylakoid membrane is permeable to ions like Mg2+^ and Cl-. The thylakoids are functional unit of chloroplast as they contain photosynthetic pigments and are the sites of light reaction. The chloroplast like mitochondria are semi-autonomous organelles.
Just like mitochondria, the evolutionary origin of chloroplast has been described by endosymbiotic theory proposed by Lynn Margulis in 1970s. The chloroplast originated from a photosynthetic prokaryote like cyanobacteria that was engulfed by a large non- photosynthetic eukaryotic cell (Fig. 2).
Fig. 2: The endosymbiotic origin of chloroplast from photosynthetic prokaryote like cyanobacteria as proposed by Lynn Margulis. Source: Author
A genome based comparison of chloroplast DNA from different plants and genome sequence of many cyanobacteria (like Synechocystis ) clearly supports the origin of chloroplast from the latter. There are also evidence to support the endosymbiotic gene transfer from the cyanobacteria to the host nucleus. The two membrane plastid is thought to be evolved from primary endosymbiosis while a secondary endosymbiosis is thought to be responsible for formation of thylakoid membrane system.
The mechanism of fission in chloroplast resembles bacteria in many aspects. In bacteria FtsZ forms a contractile ring (also known as Z ring) responsible for fission. FtsZ are GTPases, related to tubulin proteins found in all eukaryotic cells. In case of bacteria there is a single FtsZ protein responsible for forming Z ring. In chloroplast homologue of bacterial FtsZ protein are found designated as FtsZ1 and FtsZ2 which are encoded by nuclear DNA. Both play important role in forming the contractile ring responsible for chloroplast fission. It has been proposed that FtsZ1 is localized inside the chloroplast like bacterial FtsZ protein while FtsZ2 functions on the cytosolic side resulting in constriction and fission of the chloroplast into two. Another hypothesis suggests that both the Z-ring is composed of both FtsZ1 and FtsZ2 forming copolymer (Fig. 3).
Fig. 3: Chloroplast division by fission. During fission a Z-ring is assembled which is composed of FtsZ and FtsZ2 homologs of bacterial FtsZ. Source: Author
The presence of DNA in chloroplast was first reported by Ris and Plaut in 1962. Chloroplast contain double stranded, circular, naked DNA which is present in multiple copies like mitochondria. The cpDNA from many plants has been sequenced. The size of cpDNA ranges from 120-160kb and contains 60-200 genes, more as compared to mitochondria. The cpDNA contain genes for four rRNAs (4.5S rRNA, 5S rRNA, 16S rRNA and 23S rRNA), 30 tRNA, 21 ribosomal proteins, RNA polymerase, photosystem I, photosystem II, cytochrome b6f complex, ATP synthase and large subunit of ribulose1,5- bisphosphate carboxylase (Rubisco).
First the protein bound to the guidance complex interacts with Toc34 and Toc159 receptors and then is passed to the Toc75 import pore (Fig. 4). Toc34 hydrolyzes GTP which provides additional energy for protein translocation through the outer membrane and the protein is passed to the Tic complex present on the inner membrane. Another receptor Toc64 is also present on the outer chloroplast membrane and binds those proteins that have different targeting signals. The stromal side of Tic complex binds Hsp100 which hydrolyzes ATP and pulls the protein into stroma where stromal processing peptidases (SPP) cleave the transit peptide. Stromal Hsp70 are required for correct folding of the proteins.
Fig. 4: Mechanism of protein import into the chloroplast stroma. For translocation into stroma the protein should have a transit peptide. A guidance complex binds with the protein and directs it to the translocons present on the outer membrane. Source: Author
Proteins destined for the thylakoid lumen (like plastocyanin) have two different targeting sequences:
Once the protein is imported into the stroma, the stromal processing peptidases (SPP) cleave the transit peptide unmasking the second import signal known as thylakoid signal sequence or thylakoid-targeting sequence. There are three different pathways to import proteins into the thylakoid lumen:
The experimental evidences for the role of H 2 O in the formation of O 2 were given by Samuel Ruben and Martin Kamen in 1941 while working at the University of California. These workers used labeled isotope of oxygen 18 O to demonstrate that the O 2 was not produced by splitting of CO 2 but from photospliting of H 2 O.
The process of photosynthesis is essentially divided into two reactions:
1. Light-dependent reaction or light reaction: Light reaction takes place in the thylakoid membrane. The energy from sunlight is used to generate ATP and NADPH. O 2 is released during this reaction of photosynthesis. 2. Light-independent reaction or dark reaction: Dark reaction takes place in the stroma. The CO 2 fixation to produce carbohydrate using energy from ATP and NADPH takes place during dark reaction.
As already mentioned, light reaction takes place in the thylakoid membrane and is responsible for generation of ATP and NADPH. The thylakoid membrane contains light- absorbing pigments like chlorophyll and a number of membrane proteins with prosthetic groups attached to them. The light reaction can be understood in three steps:
a) Absorption of light by pigment molecules b) Release of O 2 , electron transfer and generation of proton motif force, and formation of NADPH c) ATP production
In the first step light energy is absorbed by chlorophyll molecules found in the thylakoid membrane. Light travels in packets of energy known as photons quanta, the energy which is given by Planck’s law:
E= hv = hc/λ
where h is Planck’s constant (6.626 X 10-34^ J.s c is the speed of light in a vacuum (2.998 X 10^8 m/s) λ is the wavelength of light (for visible light it is 400-700 nm)
When the photon is absorbed by a molecule, the electrons get excited to higher energy state and the molecule is said to have moved from the ground state to excited state which is an unstable state and lasts only for 10-9^ second. The electrons then return back to the ground state releasing the absorbed energy which appears in the form of either fluorescence or heat. The same phenomenon happens when a pigment molecule absorbs light photons. The most important pigment in plants is chlorophyll. The chlorophyll molecule consists of a porphyrin ring and a hydrophobic phytol tail. The porphyrin ring contains Mg2+ and functions in light absorption while the hydrophobic phytol tail keeps the pigment molecule embedded into the thylakoid membrane. Different types of chlorophyll molecules are found in different photosynthetic organisms and differ from each other in the side groups attached to porphyrin ring. Bacteriochlorophyll is present in green and purple bacteria while chlorophyll a is found to be present in all the photosynthetic organisms that evolve O 2 and is therefore universal, chlorophyll b is found in all higher plants and green
algae, chlorophyll c is found in brown algae and diatoms; and chlorophyll d is found in red algae. Apart from chlorophyll, higher plants also contain accessory pigment molecules like carotenoids which serve many important functions like light absorption, collection of excess energy from excited chlorophylls and release it as heat which otherwise lead to production of singlet oxygen which is highly reactive and can damage biological molecules.
Reaction centers and photosystems
As mentioned, chlorophyll molecules absorb light energy and convert it to the chemical energy. Several hundred chlorophyll molecules act together and form large protein complexes known as photosystems. The photosystems consist of two components: a reaction center and an antenna complex. The antenna complex consists of many light harvesting complexes (LHCs) which absorb light and pass it to the reaction center. There are two types of photosystems: photosystem I (PSI) and photosystem II (PSII). The reaction center of photosystem II is P 680 which is a chlorophyll a dimer also called special- pair chlorophylls (680 refers to the wavelength of light absorb by the chlorophyll molecule strongly). The reaction center of photosystem I is P 700. There is difference in the distribution of two photosystems in the thylakoid membranes. PSII is primarily located in the stacked grana while PSI is found in the unstacked regions. Both photosystems also differ in their function. PSII is involved in the splitting of H 2 O to produce O 2 , PSI plays important role in transferring the electrons to NADP+^ to produce NADPH.
The pigment molecules in the antenna complex of both PSI and PSII (chlorophylls and carotenoids) absorb energy of the photon and passed it to the respective reaction centers. The excited reaction centers release electron which is accepted by primary electron acceptor. This flow of electrons from photosystems may follow non-cyclic (linear) or cyclic route.
Non-cyclic (linear) electron flow
The non-cyclic electron flow also known as non-cyclic photophosphorylation involves both PSI and PSII. The PSII is a multiprotein complex consisting of more than 20 different polypeptides. Out of 20 different polypeptides, two D1 and D2 bind the reaction center P 680 and other factors involved in electron transport. The first step is the absorption of light photon by the pigment molecules of light-harvesting complex II (LHCII) of the antenna complex II. The absorbed energy is passed from antenna pigments of LHCII to the chlorophyll molecules situated inner side of the antenna complex and finally the energy is passed to the reaction center P 680. The excited reaction center passes the electrons to the primary electron acceptor like Pheophytin which is a modified chlorophyll a molecule that lacks Mg2+. The reaction center assumes a positive charge after losing electron (P 680 +) and acts as oxidizing agent (which can accept electrons). Pheophytin assumes a negative charge (Pheo-) and acts as reducing agent (can donate electrons). The Pheo-^ transfers the electron to quinone (QA) which transfers it to the second quinone (QB). The PSII also consist of a complex of four Mn2+, one Ca2+^ and one to two Cl-^ along with other proteins forming the oxygen-evolving complex (OEC) which binds two H 2 O molecules. The OEC consists of three proteins of size and 17, 23 and 33 kDa forming a complex. The OEC during extracting H+^ and e-^ from H 2 O undergoes series of cyclic oxidation states also known as S-states which are denoted as S 0 to S 4 (the S-state hypothesis was given in 1970 by Pierre Joliot and Bessel Kok). The splitting of water in the presence of light is called photolysis which releases
reduction of NADP+^ to form NADPH. The reaction center P 700 +^ is reduced by getting electron from plastocyanin. The reaction can be summarized as:
NADP+ H+^ + 2e-^ 2NADPH ------------- (2)
The overall reaction of non-cyclic electron transfer can thus be obtained combining (1) and (2):
2NADP+ 2H 2 O 2NADPH +O 2 + 2H+
The production of one molecule of O 2 thus requires four electrons from two molecules of water. The removal of one electron require one photon, so removal of four electron requires total four photons. Also to produce one NADPH two electrons are required. But due to presence of two photosystems the number of photons required is doubled (four photons are required by each photosystem). Thus eight photons are required to generate one molecule of O 2 and two molecules of NADPH. The subsequent pumping of H+^ generates proton motif force (pmf) which is harnessed to produce ATP. Thus the non-cyclic electron transport results in the generation of O 2 , NADPH and ATP.
Cyclic electron flow
In non-cyclic electron flow the electrons are passed from ferredoxin to NADP+^ to generate NADPH. The non-cyclic electron flow also produces ATP from the proton motif force generated due to subsequent pumping of H+^ to the thylakoid lumen. However, Daniel Arnon while working at the University of California, found that ATP synthesis can also take place even in the absence of CO 2 and NADP+^ suggesting that an alternative pathway does exist to produce ATP without producing NADPH. This alternative route is known as cyclic electron flow or cyclic photophosphorylation (Fig. 7).
A number of herbicides that are used to control weeds act by binding to specific component of the electron transport chain found in the thylakoid membrane. The herbicides like s-triazenes bind to the PSII reaction center and block electron transport through PSII. Some herbicides like Paraquat affect the functioning of PSI by competing with ferredoxin for electrons.
Fig. 7: Cyclic photophosphorylation. The cyclic photophosphorylation involves only PSII and generates ATP without NADPH production. Source: Author
Thus in cyclic photophosphorylation the electron from ferredoxin can be passed to plastoquinone passes the electrons to cytochrome b 6 f complex and two H+^ to the thylakoid lumen. The electrons are returned back to PSI through plastocyanin. Thus, the flow is cyclic as the electrons extracted from PSI are returned back to PSI. The cyclic electron flow thus generates proton motif force responsible for ATP synthesis but no NADPH is produced.
ATP produced by non-cyclic and cyclic photophosphorylation
As already mentioned, in non-cyclic photophosphorylation eight photons are required to generate one molecule of O2, two molecules of NADPH and 12H+^ are translocated. It has been found that 1000-2000 fold differences in H+^ concentrations exist across the thylakoid membranes. It has been proposed that translocation of 3 H+^ through chloroplast ATP synthase (also known as CF 1 -CF 0 complex) results in the generation of one ATP. So, total of 4 ATP are generated per O 2 molecule evolved or 4/8 or ½ ATP per photon absorbed. Non- cyclic photophosphorylation also results in generation of 2 NADPH. Each NADPH has free energy to generate 3 ATP molecules, so total of 6 ATP molecules are generated from 2 NADPH making a total of 10 (4+6) ATP per O 2 molecule evolved or 10/8=1.25 ATP equivalents per absorbed photon.
As compared to non-cyclic photophosphorylation, cyclic photophosphorylation seems to be more economical in terms of ATP produced only. In cyclic photophosphorylation four
Fig. 8: Calvin cycle or dark reaction. The Calvin cycle also known as C3 cycle takes place in the stroma and utilize ATP and NADPH generated during light reaction. Six CO 2 molecules react with 6 RuBP to produce 12 molecules of phosphoglycerate. Overall process requires 18 ATP, 12 NADPH to produce 12 molecules of glyceraldehyde 3-phosphate (GAP). Out of 12, 2 GAP are used for the production of sucrose in the cytosol and remaining 10 molecules are converted to 6 molecules of RuBP. The enzymes involved in the calvin cycle are- 1: ribulose 1,5-bisphosphate carboxylase or Rubisco; 2: phosphoglycerate kinase; 3: glyceraldehyde 3-phosphate dehydrogenase; 4: transketolase; 5: ribose phosphate isomerase; 6: phosphoribulose-kinase. Source: Author
The key enzyme in Calvin cycle is ribulose 1,5-bisphosphate carboxylase or Rubisco which catalyzes the conversion of ribulose 1,5-bisphosphate, a five-carbon sugar to two molecules of 3-phosphoglycerate by adding CO 2. The pathway is complex and involves number of different enzymes.
The Calvin cycle can be divided into three stages or phases:
(a) carboxylation of ribulose 1,5-bisphosphate (RuBP) to form 3-phosphoglycerate (PGA) (b) Glycolytic reversal involves reduction of PGA to produce glyceraldehyde 3- phosphate (GAP) using the NADPH and ATP (produced during light reaction) (c) Regeneration of RuBP
The stoichiometry of Calvin cycle can be summarized as:
6CO 2 + 18ATP + 12NADPH 12GAP + 18ADP + 16Pi + 12NADP+
It can be seen that dark reaction is energy consuming process where the production of 2 molecules of Glyceraldehyde 3-phosphate (GAP) requires 18 ATP and 12 NADPH molecules. Glyceraldehyde 3-phosphate (GAP) is the key product formed during dark reaction. It can be transported to the cytosol in exchange for phosphate ions (Fig. 9) or stay at the chloroplast stroma. At cytosol it is converted to sucrose (a major transport sugar in plants) in a series of reaction which is then transported into the phloem. In the chloroplast, it is converted to starch which is stored as starch granules.
Rubisco is a large large multisubunit enzyme composed of eight identical large and eight identical small subunits with molecular weight ~500kDa. Rubisco though a key enzyme in plants, has turnover number of 3 i.e. it can fix only 3 molecules of CO 2 per second. This low turnover number is compensated by increasing the number of Rubisco molecules catalyzing the reaction. It has been found that more than 50% of the protein in leaves is Rubisco and therefore it is the most abundant protein found on this planet.
Rubisco which is the key enzyme responsible for fixation of CO 2 can also act as oxygenase in a process known as photorespiration also known as C2 cycle. This seems to be an unavoidable process because of the catalytic activity of Rubisco with mild differences in the preference for CO 2 and O 2. In this process Rubisco adds O 2 to ribulose 1,5-bisphosphate forming 3-PGA and phosphoglycolate which is subsequently converted to glycolate in the chloroplast stroma. Glycolate is transported to the peroxisome where it is converted to glyoxylate and then to glycine (Fig. 10). The glycine formed in peroxisomes is transported to mitochondria and is converted to serine releasing CO 2. In this manner the fixed CO 2 is released. The process is waste of not only previously fixed CO 2 but also involves use of ATP and O 2. It has been estimated that up to 50% of fixed CO 2 can be lost due to photorespiration by crop plants growing under high light intensity.
Fig. 10: Photorespiration or C2 cycle. The pathway involves three organelles: chloroplast, peroxisomes and mitochondria. The 2-phosphoglycolate formed due to oxygenase activity of Rubisco is metabolized in these organelles. There is lost of already fix CO2 and therefore the process is not beneficial for plants. The C2 cycle involves following enzymes- 1: Rubisco; 2: phosphoglycolate phosphatase; 3: glycolate oxidase; 4: serine:glyoxylate aminotransferase; 5: glycine decarboxylase, serine hydroxymethyl transferase; 6: serine:glyoxylate aminotransferase; 7: hydroxypyruvate reductase; 8: glycerate kinase. Source: Author
Photorespiration is more prominent in a hot dry environments where in order to prevent water loss plants close their stomata. This results in decrease in the concentration of CO 2 promoting oxygenase activity of Rubisco. Therefore, the crop plants that grow in hot dry environmental conditions like sugarcane, sorghum, crabgrass and corn have developed mechanisms to avoid this loss of fixed CO 2. These crop plants have evolved a two-step mechanism which is known as C4 pathway as the first stable compounds formed using radioactive 14 C (reported by Hugo Kortschak in 1965) were found to be a four Carbon compounds like oxaloacetate and malate. The complete C4 cycle was elucidated in 1960s by Marshall Hatch and Rodger Slack and therefore it is also known as Hatch and Slack pathway. The plants that employ C4 cycle also known as C4 plants continue photosynthesis at even at very low CO 2 concentration and have characteristic leaf anatomy. The leaves consists of single layer of bundle-sheath cells surrounded by mesophyll cells. The mesophyll cells lack Rubisco in their chloroplast and fix CO 2 using enzyme PEP (phosphoenolpyruvate) carboxylase (Fig. 11) forming oxaloacetate. The enzyme PEP carboxylase is found exclusively in C4 plants. The oxaloacetate is converted to malate which is transported to bundle-sheath cells where it is decarboxylate to produce CO 2 and pyruvate. In this way the CO 2 is concentrated which enters into the Calvin cycle. The bundle-sheath cells have CO 2 concentration as high as 100 times that of mesophyll cells.
