Photosynthesis

This slideshow explains the details about Photosynthesis process. It has covered all the aspects such as definition, significance, photosystems, Hill reaction, Calvin cycle, HSK cycle, CAM pathway, Photorespiration, etc. of photosynthesis. This slide show will be useful to College students and the students who are appearing for various competitive examinations. .This slide show is equally beneficial to the students who want to pursue career in the biological sciences.

1. Photosynthesis Dr. Anil V Dusane Sir Parashurambhau College Pune [email protected] www.careerguru.co.com 1

2. Introduction of Photosynthesis This is the most important physico -chemical process. The existence of life on the earth is dependent on the photosynthesis. Definition: It is physico -chemical process in which green cells fix (convert) energy of sunlight into chemical form with the help of CO 2 , water and chlorophyll pigment. Photosynthesis is the physiological process that converts the radiant energy into chemical energy and the chemical energy is stored in the form of organic food material. 6CO 2 + 12H 2 O----------------------------------------C 6 H 12 O 6 + 6H 2 O+ 6CO 2 sunlight and chlorophyll pigments 2

3. Importance of photosynthesis Source of energy for all the living activities: To carry out all the living activities in the autotrophic and heterotrophic organisms the solar energy captured by the process of photosynthesis. About 200 billion tonne of sugar produced and about 10 17 KJ energy is captured by this unique process. Balance of CO 2 and O 2 in air : Every year green plants produce about 500 billion tonne of O 2 by this process. Thus, O 2 and CO 2 balance in the air is maintained by respiration and photosynthesis as these processes are exactly opposite to each other. Raw material for industrial products : The products such as textile fibres , timber, pulp, cellulose, alcohol, tanning gum, resins, etc. are ultimately derived from photosynthesis process. Renewable and non-renewal energy sources: Biomass, alcohol. biogas, etc. are the ultimate products of photosynthetic activity. 3

4. Photosynthetic apparatus Photosynthetic apparatus of higher plants is present in chloroplast which is the site of light and dark reactions of photosynthesis. The light absorbing pigments (chlorophylls) of thylakoid membranes are arranged in functional sets (arrays) called photosystems . In spinach chloroplasts, each photosystem contains about 200 molecules of chlorophylls and about 50 molecules of carotenoids. Almost all the pigment molecules in photosystems absorb photons. These are called light harvesting or antenna molecules. Light absorbed by hundreds of antenna molecules are then transferred at extremely high rate to a site at which chemical reaction occurs. This site is called reaction center . 4

5. Photosynthetic reaction centre Only few chlorophyll molecules those at reaction center mediate the transformation of light energy into chemical energy. Chlorophyll molecules at the reaction center are chemically identical with other chlorophyll molecules in photosynthetic unit. The only difference is that the energy level of their excited state is lower than that of other chlorophylls, which makes them energy trappers. 5

6. Photosynthetic unit- Quantasomes Quantasomes represent photosynthetic unit. The photosynthetic unit is the smallest group of photosynthetic pigments and other associated compound, which are necessary for the photochemical act. The photochemical act involves the tapping of light and excitation of electrons. These are arranged in monolayer on thylakoid. Size of quanta: 18X17X10 nm (smaller than ribosomes). It is with a molecular weight of about 2 million. Internally the quantasome is differentiated into two parts i.e., peripheral region (enriched with pigment molecules) and the middle region or energy center . 6

7. Composition of Q uantasomes 230 chlorophyll molecules (160 chl a, 70 chl b), 48 molecules of carotenoids, 47 quinones, 115 phospholipids, 144 diagalactosyldiglyceride , 346 monogalactosyldiglyceride , 48 sulfolipid , S terols and unidentified lipids. In these specialized quanatasome structures, only a single molecule of pigment can absorb energy at a time and all the other molecules support the action of light reaction of photosynthesis 7

8. Quantasomes 8

9. Photosynthetic pigments Chlorophylls are the complex structures consisting of four pyrrole rings around a central Mg ++ atom and a long hydrocarbon chain of phytol. It is a derivative of porphyrin. Chlorophyll is the most efficient light absorbing pigments as it contains extensive network of conjugated double bonds (polyenes) that gives capacity to absorb visible light and transfer excitations. Chlorophylls and carotenoids are important photosynthetic pigments. 9

10. Types of Photosynthesis pigments Chlorophyll a ( C 55 H 72 O 5 Mg) is the principle element of energy trapping. Chlorophyll b ( C 55 H 70 O6Mg ): It occurs in green and higher plants absent in BGA, red algae, brown algae and diatoms. Chlorophyll C: Abundant in diatoms and brown algae. Chlorophyll d: Abundant in red algae. Carotenoids: are protective against photooxidation of chlorophylls and absorb light energy of the middle of visible spectrum and transfer it to Chlorophyll a. These are categorized as carotene and xanthophylls. Carotene ( C 40 H 56 ) is orange in colour . B-carotene is found in higher plants and most of algal species, Xanthophylls ( C 40 H 56 O 2 ) are a Oxygen derivative of carotene. Phycobilins are accessory pigments distributed only in Cyanophyta and red algae. There two types - Phycoerythrin (red) and phycocyanin (blue). 10

11. Red drop and Emmerson effect Emmerson and Lewis (1943) worked on the quantum yield of photosynthesis. They found that an average quantum yield is 12% and this quantum yield decline sharply at the wavelength greater than 680 nm (in red zone). The decline in quantum yield in red zone of spectra, called red drop . The work of Haxo and Blinks (1950) on Porphyra nereogitis (red alga) also confirmed the phenomenon of red drop. Emmerson and Chalmers (1951) observed that the red drop can be cancelled by simultaneously providing shorter wavelength. They also noted that the effect of two wavelength (shorter and long wavelength) simultaneously on the rate of photosynthesis exceeds the sum effect of both wavelengths uses separately. This enhancement of photosynthetic rate under influence of two wavelengths (long and short) of light is called Emmerson effect. 11

12. Red drop and Emmerson effect 12 Emmerson effect = Rate of release of O2 under both wavelengths – Rate of release of O2 in short wavelength (red)/ Rate of evolution of O2 under long wavelength (far red)

13. Significance of Emmerson effect This effect had made radical change in the understanding of the mechanism of light reaction. For the first time he claimed that two photosystems viz. Photosystems I and II occur in chloroplast. Photosystem-I contains the long red wavelength absorbing form chl a 683 (P700) while photosystem system II contains short red wavelength absorbing form Chl.a 673 (P680). Emmerson also claimed that both the photosystems work harmoniously and responsible for photolysis of water and reduction of NADP. The photosystem-I is under the influence of long wavelength and photosystem II is under the influence of short wavelength and that is why the two wavelengths (short and long) enhance the photosynthesis rate. 13

14. Photosystems A photosystem is the assemblage of 200-400 pigment molecules together with a primary electron acceptor and a series of electron carriers. The photosystems are located within thylakoid membrane. The function of photosystems is to capture the light energy for photosynthesis. Based on the type of photochemical reaction center, a set of antenna molecules and electron carriers photosystems are categorized into viz. PSI and PSII 14

15. Photosystem-I This is called as photosystem-I because it was discovered first. The reaction centre of PS-I is designed as P700 (because chl.a absorbs light wavelengths that are of P700 nm (far red). This photosystem has a high ratio of chl a to chl b. Composition of PSI: 200 Antenna chlorophylls 50 Carotenoids 1 P700 reaction centre 1 Bound ferrodoxin 1 Soluble ferrodoxin 1 Cytochrome b 563 cyt b 6 1 Cytochrome C 552 or cyt f 1 Plastocyanin 1 Ferridoxin -NADP-reductase. 15

16. Mechanism of Photosystem-I Antenna molecules first capture the light. The absorbed energy is sent to (moves to) P700 by resonance energy transfer mechanism. The excited reaction centre (P700) loses an electron to an electron acceptor and becomes P700 + . This is now a strong oxidizing agent. It quickly acquires electron from plastocyanin. Electron from P700 is transferred to ferrodoxin (bound) and then to soluble ferredoxin. The reduced ferrodoxin transfers its electron to NADP + to form NADPH. The conversion of NADP + to NADPH is catalyzed by ferrodoxin –NADP reductase . 2 ferrodoxin (reduced) + H + + NADP + ------ ( ferrodoxin -NADP reductase ) + 2 ferrodoxin (oxidized) + NADPH. Importance: PSI generates NADPH via reduced ferrodoxin and light absorption by PSI creates a powerful reducing agent. 16

17. Photosystem II I t is discovered after photosystem I. Composition of PSII. 200 Antenna chlorophylls (a and b) 50 carotenoids 1 P680 reaction centre 1 Primary electron donor (Z) 1 Primary electron acceptor (Q) 4 Plastoquinone 6 Manganese atoms 2 Cytochrome b 6 . 17

18. Photosystem II It consists of 3 pigments-chlorophyll a, b (c or d according to species) and carotenoids. The reaction centre molecule ( Chl a) is called P680 because it absorbs wavelengths that are 680 nm (red) and less. The primary electron acceptor of PSII is substance Q (of unknown molecular weight.). The other carriers in order of decreasing free energy are Plastoquinone (PQ), Cyt f , and PC. The electron flow through PSII is as follows P680→Q→PQ→Cyt→PC. It is during PSII oxygen is evolved. Photosystem II generates a strong oxidant that splits water. The redox potential of this reaction centre is about + 0.8 V. 18

19. Mechanism of Photosystem II The electron from the photoactivated reaction centre of PSII is transferred to Q (which is tightly bound molecule of Plastoquinone). The electron is then transferred from Q to a pool of mobile plastoquinones and then to cyt b559 (b6). This electron is then transferred to Cytc 552 (formerly called cyt f). The final electron carrier from PSII to PSI is plastocyanin (which contains Copper). Cu atom facilitates electron transfer as Cu alternates between +1 and +2 oxidation states. The electron transfer from reduced plastocyanin to oxidized form of P700 enables this reaction centre to again serve as an electron donor to form NADPH when illuminated. The overall reaction initiated by light in PSII is 4 P680 + 4H + + 2 QB + light (4 photons) ----- 4 P680 + + 2 Q 2 H 2 . The water splitting activity is an integral part of PSII reaction centre . 19

20. Coordination between PSI and PSII According to Junge (1982), each granum has 200 units of PSI and PSII. The PSI and PSII functions in close co-ordination because they jointly function to transfer the electrons from water to NADP. Since these systems are located a little apart that is why certain intermediates (carriers) help to carry electrons from PSI to PSII. These carriers are found to be of two types. i ) Plastocyanins (copper containing proteins) in its reduced form can carry an electron form PSII to PSI. ii) Plastoquinones (group of quinones) carry two electrons and two H+ from PSII to PSI. Besides the above additional components in the form of cytochromes occurs in thylakoids Cyt b6 and Cyt f in between PSI and PSII and help in electron transport between them. Cyt b3 and Fd ( FeS protein) also participate in the light mediated electron transport. Electron flow accompanied by proton pumping across the thylakoid membrane and the proton motive force thus created drives ATP synthesis. 20

21. Light reaction It produces ATP and NADPH. Hill reaction: Robert Hill (1939) discovered that the isolated chloroplasts evolve oxygen in the presence of water, light and a suitable electron acceptor (ferricyanide). He recorded that the reduction of ferricyanide to ferrocyanide takes place as follows: 2H 2 O + 4 Fe +++ --------- (illuminated chloroplast) O 2 + 4H + + 4 Fe ++ Hill reaction has clearly shown that O 2 evolution takes place in the absence of CO 2 . Hill used non-biological hydrogen acceptor, a dye-2,6 dichlorophenolin-dophenol now known as Hill reagent . Which is in oxidized form (A) is blue and its reduced form (AH 2 ) is colourless . When leaf extracts supplemented with the dye and illuminated, the blue dye become colourless and O 2 was evolved. This was the first clue, which indicated that how the absorbed light energy is converted into chemical energy. Hill found that CO 2 was not required for this reaction. 21

22. Diagrammatic representation of Hill reaction 22

23. Hill reaction The hill reaction represents light reaction in photosynthesis. 2H 2 O + 2A---------------(light, chloroplast) 2AH 2 + O 2 . artificial hydrogen acceptor (oxidized form) and AH 2 - reduced form. After Hill’s experiment, Arnon (1951) showed that NADP + serves as electron (hydrogen) donor and plays significant role in the reduction of CO 2 . The Hill reaction may be expressed as 2NADP + + 2H 2 O---------------------(light, chlorophyll) 2NADPH +2H + +O 2 NADP + (Nicotinamide Adenine Dinucleotide Phosphate) is reduced to form NADH For every O 2 molecule liberated or for every molecule CO 2 reduced, at least 2 molecules of NADPH are produced. Besides NADPH, ATP molecules are also formed through phosphorylation. 23

24. Significance of Hill reaction Hill reaction is considered as a landmark in elucidation of the mechanism of photosynthesis due to following reasons: It has shown that oxygen release can occur without reduction of CO 2 . (The artificial electron acceptors such as ferricyanide can substitute for CO 2 ). It confirmed that evolved oxygen comes from water and not from CO 2 (as CO 2 was not present). Use of radiotracers 18 O later proved this. It showed that the isolated chloroplasts could perform a partial photosynthesis. It revealed that a primary event in photosynthesis is the light activated transfer of an electron from one substance to another against chemical potential gradient. The reduction of ferric to ferrous ion by light gives a clue for the conversion of light energy into chemical energy. This reaction shows that the source of oxygen is water, and this water undergoes splitting (photolysis). The splitting of water releases electrons and these are required for reduction of CO 2 . The splitting of water, H 2 and O requires light and chlorophyll 24

25. Photophosphorylation Arnon (1954) showed that isolated chloroplast could produce ATP from ADP and Pi in the presence of light. Arnon termed the formation of ATP molecules as ‘photophosphorylation’ (as mitochondrion is not involved). 2ADP + 2 Pi + 2 NADP + 4H 2 O ------ (light, chloroplast ) 2 ATP + 2NADPH 2 + 2 H 2 O + O 2 . Definition of phosphorylation: Chlorophyll molecules when excited by sunlight, energy rich electrons are emitted, these then transferred through a series of electron carrier in a stepwise manner. During this sequential transfer of electrons, the energy of electrons is lost bit by bit and the lost energy is picked up by ADP and Pi molecules to synthesize energy rich molecule (ATP). This process of synthesis of ATP molecules due to light energy is known as photophosphorylation. ATP produced (by chemosynthesis mechanism) in above process is used up for CO 2 assimilation (fixation) in dark reaction of photosynthesis. There are two pathways of electron transfer (photophosphorylation) viz. Cyclic photophosphorylation (cyclic electron transfer) and non-cyclic photophosphorylation (non-cyclic electron transfer). 25

26. Cyclic photophosphorylation The electrons released from chlorophyll (P700) returns (get back) in a continuous chain again to chlorophyll. In this the electron travels in a cyclic fashion hence named as cyclic phosphorylation. Salient features: Arnon and his associates established the steps of cyclic photophosphorylation. This takes place in presence light and chlorophyll. This mechanism is possible only when PSI (P700) participates. Substrates required are ADP and Pi. The products are ATP and H 2 O. Water does not participate hence there is no evolution of O 2 There is no formation of reduced NADPH molecule. Only ATP is produced. In this process no outside electron donor is needed. The flow of electron is PSI—FRS— Fd --- Cyt b 6 --- Cyt f ---PC and PSI sequence. Overall reaction is ADP + Pi------------------------(light, chlorophyll) ATP + H 2 O 26

27. Mechanism of cyclic phosphorylation 27

28. Significance of cyclic phosphorylation ATP formed during in any one type of photophosphorylation (including cyclic) is insufficient for the fixation of CO 2 during carbohydrate formation. Therefore, cyclic and non-cyclic must work harmoniously. 28

29. Non-cyclic phosphorylation An electron released from chl a does not returns to chl.a hence this process is called non-cyclic photophosphorylation. This is also called Z scheme electron transport because the electron travels in a zig zag fashion (not in a cyclic manner). Salient features: Flow of electron is unidirectional This is the major pathway of light reaction involving both, PSI and PSII. Oxygen produced or ADP phosphorylated is proportional to the amount of NADP + present in the system and this process goes on until all NADP + is reduced. During this process quanta causes photolysis of water molecule. It is broken down to two electrons, two protons and one oxygen atom. There is evolution of oxygen in this process electrons are provided by water molecules (tracer technique has shown this). H 2 O--- 2e - + 2H + + O. 29

30. Non-cyclic phosphorylation 6. Electron flow normally takes place along electrochemical gradient i.e. from more electron negative components to more electrons positive one. However, in this the electron transport from pigment II to compound Q takes place against electron gradient. Chl. (PSII)---Q—PQ-- Cyt b 6 — Cyt f ---PC----PSI---FRS-----( Fd.UQ )--- Fd ------NADP + 7. The two electrons from water replace two electrons that have left the reaction centre (P680) and PSII. 8. In this process two molecules of ATP are formed per two molecules of NADP + reduced or one molecule of oxygen evolved, or two molecules of water oxidized. 9. During sequential transfer of electrons an ATP molecule is produced in between Cyt f and plastiquinone . Over all reaction is 2ADP+2Pi+2NADP + +2H 2 O-----(light) 2 ATP +2 NADPH +H + +O 2 . 30

31. Non-cyclic photophosphorylation 31

32. Mechanism of Non-cyclic photophosphorylation Light (photon) absorbed by PSI (P700) result in the excitation of one of electron from chlorophyll molecule. This excited electron is picked up by FRS ( Ferrodoxin Reducing Substance) and then finally goes to ferrodoxin . The reduced ferrodoxin transfers its electron to NADP + to form NADPH (in presence of ferrodoxin –NADP reductase). However, PSI remains in the excited state, as it has not got back its electron. Some electrons are used for reduction of NADP + whereas others is passed to Cytf via Cyt b 6 . Thus Chl.a remains in an unstable state since there is deficiency of electron due to photoreduction. 32

33. Mechanism of Non-cyclic photophosphorylation Meanwhile PSII (P680) absorbs light energy and ejects one of its electrons. This excited electron is picked up by plastoquinone. An electron produced in the photolysis of water (H 2 O-------2e - + 2H + +O) replaces the electron lost by PSII. Plastoquinone accepts electron and then it gets reduced (plastoquinone + 2H ===reduced plastoquinone). The reduced plastoquinone then donates the electron to Cyt b 6 . The electron is transferred to cytochrome f then plastocyanin and P700 (PSI). P700 can accept an electron only if it has lost one of its own when excited by the energy in second photon of light. In above process excess electron energy is used for the synthesis of ATP (from ADP + Pi). ATP formation takes place in between Cyt b 6 and cyt f. 33

34. Cyclic photophosphorylation Vs Non-cyclic photophosphorylation Cyclic photophosphorylation Non-cyclic photophosphorylation 1 Expelled (excited) electrons returns to chlorophyll Expelled (excited) electrons do not return to the chlorophyll. 2 Only PSI functions Both PSI and PSII functions 3 In bacteria only cyclic photophosphorylation occurs In green plants both cyclic and non-cyclic occurs. 4 No evolution of O 2 as water does not participate O 2 is evolved as water participates 5 Energy is captured in form of 1 ATP. Energy captured inform of ATP and NADPH 6 Electron flow- chl (PSI)---FRS— Fd — Cyt b 6 — Cyt f—PC— Chl (PSI) PSII—Q--PQ--- cytf --PC---PSI—FRS— Fd —NADP + 7 Not inhibited by DCMU (Dichlorophenyl-dimethyl urea) Stopped by DCMU 34

35. Electron carriers These maintain an optimal geometry for the energy transfer between chlorophylls. Quinone (Q): When photosystem II is excited that time e - is transferred to primary electron acceptor Plastoquinone (PQ)- Group of quinones. PQ + 2H ===reduced plastoquinone. The reduced PQ then donates its e - to cyt b 6 (these also carry 2e - and 2H + from photosystem II and I. Cyt b 6 ( Cyt b559): Proteins are associated with Fe i.e. haeme . Its Redox potential is near to 0. Its stereoscopic properties are similar to that of Cyt b of mitochondria. Cyt f: It is a c type of cytochrome called Cyt c 552/c554. It is insoluble membrane bound protein. Plastocyanin PC: A copper protein. It is a soluble copper containing e - transfer protein. It is having 2 Cu atoms, which are the sites of oxidation-reduction. PC is blue in oxidized state and colourless in reduced state. It is likely that this may be immediate e - donor to P700. FRS ( Ferrodoxin Reducing Substrate): These compounds are also designated as X or Z. From photosystem-I e - are accepted by FRS. 35

36. Dark reaction This can take place either in absence or presence of light. The overall photosynthetic rate is regulated by the light reactions. Light does not have any direct effect on this process. This is also known as carbon assimilation phase the carbohydrate synthesis takes place. In this reaction products of light reaction viz. ATP, NADPH is used to reduce CO2 into glucose. The use of CO2 isotopes helped to trace the secrets of the dark reaction. CO2 is fixed by C3 pathway (Calvin cycle), C4 pathway (Hatch Slack cycle) and CAM (Crassulacean Acid Metabolism). Blackmann (1905) claimed that light is not necessary for reduction of CO2. 36

37. Calvin cycle Malvin Calvin (1953) for the first described path of carbon in photosynthesis. Calvin was awarded with a Noble Prize for this work in 1961. Chromatography and radioautography helped Calvin to trace the path of carbon in photosynthesis. Salient features: It has been named variously such as Calvin cycle, Bassham and Calvin cycle, Carbon assimilation, Blackmann reaction, Calvin–Benson cycle, etc. It is also known as C 3 pathway because the product of first reaction contains three carbon atoms. It occurs in stroma (as it contains most of the enzymes required for CO 2 fixation). In this CO 2 combines first with Ribulose diphosphate ( RuDP ) to form two 3-carbon molecules of phosphoglycerate. 18 ATP molecules are required for the synthesis of one glucose molecule. The overall efficiency of photosynthesis under standard conditions is at least 30%. Net reaction for Calvin cycle: 6CO 2 +18 ATP+12 NADPH+12H 2 O---- C 6 H 12 O 6 +18 ADP+18 Pi+12 NADP + +6H + 37

38. Steps in Calvin cycle Carboxylation, R eduction of (PGA), F ormation of fructose 6-phosphate and Regeneration of acceptor ( RuDP ). Carboxylation: It is the first reaction in which CO 2 combine with 5-carbon acceptor i.e., Rubulose1,5 biphosphate (RuBP). An unstable 6C compound is immediately breaks into two molecules of a 3-carbon acid i.e., Phosphoglyceric acid (PGA). Here the first product of CO 2 fixation has 3C atoms that is why Calvin cycle is also referred as C 3 pathway. The reaction is catalyzed by Ribulose biphosphate carbosylase /oxygenase (RUBISCO) Ribulose1-5 disphoshpate (5C)+CO 2 (1C)+H 2 O ==(Rubisco) 3-PGA (2 molecules, 3C + 3C) + 2H + 38

39. Steps involved in Calvin cycle Reduction of PGA: Conversion of 3-phosphoglycerate to glyceraldehyde-3-phosphate takes place. This requires a large input of energy. Each PGA is phosphorylated by ATP and reduced by NADPH to form PGAL. PGA + NADPH + H + --------- ( ATP ) PGAL. This reaction takes place in two sub steps. i ) 3 phosphoglycerate kinase is in the stroma, and it catalyzes the transfer of phosphate from ATP to 3-PGA and yields 1,3 diphosphoglycerate. PGA + ATP --------- ( phosphoglycerokinase ) 1,3-diphosphoglyceric acid + ADP. ii) 1,3-diphosphoglycerate+NADPH+H + -------( glyceraldehyde3-phosphate dehydrogenase ) 3 PGAL+NADP NADPH donates electrons and reduction is catalyzed by glyceraldehyde –3-phosphate dehydrogenase producing glyceraldehyde 3-phsophate. The ATP and NADPH required in the above reaction come from the light reaction. Five out of every six molecules (5/6 th ) of this sugar combine with 3 ATP molecules to form 3 molecules of RuDP (which is used up to pick up more carbon dioxide) and one out of every six molecules (1/6 th ) used to synthesize more complex end products (sugars). It takes six turns to produce two PGAL molecules that from one glucose molecule. The 5/6 of total PGA produce a number of 4C, 7C and 5C sugars by series of intermediate reactions one of the 5C intermediates is ribulose-5-phosphate and it reacts with ATP resulting regeneration of ribulose-1-5 diphosphate. 39

40. Steps involved in Calvin cycle Formation of Fructose-6-phosphate: 3PGA to Fructose 6- phosphate. Glyceraldehyde 3-phosphate + Dihydroxyacetone phosphate (DHAP) condenses in the presence of aldolase . Generation of RuDP : i )Fructose-6-phosphate (C6) + glyceraldehyde-3-phosphate (C3) ---- ( transketolase ) xylose-5-phosphate (C5) + erythrose 4-phosphate (C4). ii)Erythrose 4-phosphate (C4) + DHAP (C3) ------ ( aldolase ) Sedoheptulose , 1,7 diphosphate. iii) Sedoheptulose , 1,7 diphosphate (C7) + glyceraldehyde 3-phosphate (C3)-------( transketolase) ribose-5-phosphate (C5) + xylulose-5-phosphate (C5). iv) Phosphopentose epimerase converts xylulose 5-phosphate into ribulose-5-phosphate. v)Xylulose 5-phosphate--------- ( phospho pentose epimerase ) ribulose 5-phosphate.   40

41. Steps involved in Calvin cycle vi) Phospho pentose isomerase converts ribose-5-phosphate into ribulose-5-phosphate. vii)Ribose-5-phosphate ----------( phospho pentose isomerase ) ribulose-5-phosphate. viii) Phospho ribulose kinase catalyzes phosphorylation of ribulose-5-phosphate to regenerate ribulose1,5 diphosphate (which is CO 2 acceptor). Ribulose 5-phosphate+ATP----( phospho ribulose kinase ) ribulose 1,5 diphosphate + ADP+ H + Sedoheptulose 1, 7 diphosphate ----- ( phosphatase ) Sedoheptulose 7-phosphate. If all the six molecules of RuDP combine with 6 molecules of CO 2 and form 6 molecules of glucose, the cycle cease to operate and for the next turn there will be no RuDP to accept CO 2 molecules. Thus, for entry of every 6 CO 2 molecules one goes for hexose formation and rest go for the regeneration of RuDP . 41

42. Steps involved in Calvin cycle Formation of sugars: The PGAL molecule (1 out of 2) undergoes isomerization to form a molecule of DHAP the enzyme triose phospho isomerase catalyzes this reaction. 3 PGAL --------- ( triose phospho isomerase ) DHAP 3 PGAL + DHAP ( aldolase) ----fructose 1, 6 diphosphate. Fructose-1-6 diphosphate gets converted into 6-mono phosphate due to the loss of one phosphate group under the influence of phosphatase . Fructose 1,6 diphosphate------ ( phosphatase , H 2 O) Fructose-6-monophosphate (FMP). FMP isomerases into glucose- mophosphate under the influence of isomerase FMP -------- ( isomerase) Glucose-6-monophosphate (GMP). FMP or GMP undergoes dephosphorylation to form either fructose or glucose. These monosaccharides unite to form sucrose and various other carbohydrates For every 3 CO 2 molecules fixed, one molecule of triose phosphate (PGAL) is produced, and 9 ATP and 6 NADPH are consumed. 42

43. Calvin cycle 43

44. Overview of Calvin cycle 44

45. Significance of Calvin cycle CO 2 is absorbed and fixed to form carbohydrates i.e., starch. ATP and NADH 2 are used and are again converted into ADP and NADP for reuse in light reaction. RuDP is regenerated and again ready for picking CO 2 molecules. An enzyme, RuDP carboxylase catalyzes the joining of CO 2 with RuDP or O 2 with RuDP . The two molecules are alternative substrates that compete with each other for the same active site of enzymes. The more abundant of two molecules gets occupy the active site and subsequently joins with RuDP . When the concentration of CO 2 in stroma is higher as compared to O 2 concentration then CO 2 joins with RuDP and Calvin cycle is initiated. When reverse the case CO 2 joins with RuDP and a pathway known as photorespiration is initiated. 45

46. HSK/C4 pathway/B-carboxylation It has been named variously such as Hatch, Slack and Kortschak or B-carboxylation or Bicarboxyllic acid Cycle or Co-operative photosynthesis or C4 pathway, etc. It is known as C4 pathway because the first product formed contains 4C atoms. For the long time it was thought that C3 pathway is the only pathway for the fixation in all green plants. Kortschak et al (1954) discovered an alternative pathway for CO2 fixation in photosynthesis. Kortschak found that the first product formed was 4C compound in sugarcane. Later MD Hatch and CR Slack (1966) confirmed it in several plants . 46

47. HSK/C4 pathway/B-carboxylation The pathway occurs in majority of grasses viz. Panicum maxicum , Artiplex spongosa , Chloris guyana , etc, also in the plant like Maize. Besides Graminae it occurs in plants belonging Euphorbiaceae , Portulacaceae , Cyperaceae , Amaranthaceae , Composiatae , Chenopodiaceae, etc. However, C4 pathway does not occur in all graminaceous plants e.g., wheat, rice, etc. 47

48. Structural (anatomical) peculiarities of C4 plants Plant with C 4 pathway has several unique features known as Kranz anatomy (in German Kranz = wreath). The Kranz anatomy is characterized by the lack of differentiation of mesophyll into palisade and spongy parenchyma. In this, vascular bundles are surrounded by a layer of distinct parenchyma cells, which are arranged radially. These constitute bundle sheath. In transverse section of vascular bundle, the bundle sheath appears like a wreath hence the name Kranz anatomy. In addition to this, there are two distinct kinds of photosynthetic cells i.e.bundle sheath cells and mesophyll cells. The chloroplasts in bundle sheath cell are large but with poorly developed grana. Mesophyll cells with small chloroplasts and having well developed grana. 48

49. Kranz anatomy 49

50. Mechanism of HSK Phosphoenol pyruvate (PEP) is the initial acceptor of CO 2. Phosphoenol pyruvate carboxylase is found abundantly in mesophyll cells. This catalyzes the condensation of CO 2 with PEP and formation of 4C compound i.e. Oxaloacetic acid (OAA) takes place. The reaction requires the participation of water. PEP+CO 2 (1C) + H 2 O ----( PEP carboxylase ) OAA (4C) + H 3 PO 4 (phosphoric acid) The function of PEP carboxylase is similar to Rubisco. OAA formed is relatively unstable and it readily gets converted into either malic acid or aspartic acid. OAA is reduced to malic acid by light generated NAPH 2 . The reaction is catalyzed by malate dehydrogenase . OAA + NADPH + H + ----------- ( malate dehyrogenase ) Malic acid + NADP + . The malic acid is transferred to the chloroplast of bundle sheath cells. 50

51. Mechanism of HSK In bundle sheath cells, malic acid undergoes oxidative decaboxylation and pyruvic acid is formed. This reaction is catalyzed by malate dehydrogenase . Here the malic acid is decarboxylated. Malic acid+NADP + ------- ( malate dehydrogenase ) Pyruvic acid+ NADPH+H+CO 2 . NADPH formed in above reaction travels back to mesophyll cells to regenerate malic acid. Pyruvic acid also goes in mesophyll cells where it utilized the light generated ATP to produce PEP again. Pyruvic acid +ATP+ H 3 PO 4 ----------- ( pyruvate phosphate dikinase ) PEP + Pi. The release CO 2 (formed due to the oxidative decarboxylation of malic acid) enters the Calvin cycle in the usual way by condensing with RuBP. The Calvin cycle operates in bundle sheath cells. In this cycle, the carboxylation takes place at two sites therefore this pathway is also known as ‘decarboxylation pathway’. 51

52. HSK pathway/C4 pathway 52

53. Biological significance of HSK pathway This pathway is considered one of incredibly significant pathway. Adaption to the tropical climate: The real significance of C 4 pathway is the adaption to a tropical climate. In tropical climate, temperature is high, so stomata are partially opened so less CO 2 is available for photosynthesis. This cycle makes more efficient use of CO 2 and increases the uptake of CO 2 though low-level CO 2 is present. This pathway is efficient even at 10 ppm concentration of CO 2 . High temperature tolerance: CO 2 fixing enzyme i.e., PEP carboxylase is insensitive to high temperature while RuBP carboxylase is sensitive to temperature. The plants lacking C 4 pathway lose 25% - 50% of their fixed carbon by photorespiration. The plants with C 4 pathway have little photorespiration because the oxygenation of RuBP is inhibited by high concentration of CO 2 in their bundle sheath cells. The carboxylation in bundle sheath increases CO 2 concentration. This CO 2 is used in C 3 pathway. 53

54. Biological significance of HSK pathway E conomical use of water: Less water is lost through C 4 plants because their stomata are partially close. So, the plants release less water during CO 2 fixation as compared to C 3 plants and thus make more economical use of water. Through this pathway nature has experimented with variation and has provided alternative pathway for the fundamental process like photosynthesis. To have only one pathway for the process like photosynthesis is catastrophic. Thus, the nature has provided some variation through this pathway. 54

55. Distinguish between C3 pathway and C4 pathway Characteristics C 3 pathway C 4 pathway CO 2 acceptor RuBP PEP First product PGA ( 3C) OAA (4C) Chloroplast Normal Dimorphism Photosystem PSI and PSII. Bundle sheath cell lack PSII. Enzymes for C 3 In mesophyll cells In bundle sheath cells. CO 2 compensation of photosystem 50-150 ppm 0-10 ppm Photorespiration Present Absent or negligible Temperature (optimum) 10-25 o C 30-45 C Pathways involved Only C 3 Both C 3 and C 4 High rate of O 2 Inhibits photosynthesis No effect on photosynthesis Number of ATP required to synthesize 1 glucose molecule 18 ATP 30 ATP 55

56. Crassulacean Acid Metabolism (CAM) This is one of the pathways of CO2 fixation (other than C3 and C4 pathways). This is called as CAM because it was first recorded in Crassulaceae family. About 2,000 species show CAM mechanism for CO2 fixation. This pathway is quite common in many of the plants belonging to Crassulaceae, Cactaceae , Orchidaceae , Bromeliaceae , Asclepediaceae , Euphorbiaceae etc. Many orchids and bromeliads that grow as epiphytes also show CAM pathway. 56

57. Important features of CAM pathway CAM plants keep their stomata open mainly at night and close during day yet achieves net fixation of CO2. During daytime CO2 uptake is negligible. Most of the CAM plants grow in acidic habitat and have exceptionally low rates of transpiration and with succulent habit. CAM plants fix atmospheric CO2 mainly at night when stomata are open, but they cannot use the Calvin cycle because this operates only in the light. These plants have a diurnal fluctuation of organic acid, some degree of succulence and large storage vacuoles. 57

58. Important features of CAM pathway In CAM plants, initial CO2 fixation and the Calvin cycle operate at different times but in the same cells in contrast to C4 plants where they operate at the same time but in different cells. There is limit to the amount of malic acid, which can be stored. This usually, determines the overall photosynthetic capacity. An efficiency of CAM is less as compared to C4 plants. But this is the price paid for the conservation of water. Metabolic pathway of CAM is similar to C4 pathway in many respects. However, it is much more flexible photosynthetic strategy than C4 pathway 58

59. Mechanism of CAM (Night time) Stomata are open, CO 2 is fixed through the action of PEP carboxylase to malic acid i.e., this acid is formed at the night by carboxylation of phosphopyruvic acid in the presence of enzyme PEP carboxylase . PEP + CO 2 ------------- ( PEP carboxylase ) OAA Later O.A.A. gets converted to malic acid with help of malate dehydrogenase . OAA + NADPH ---------- ( malate dehydrogenase ) malic acid + NADP + This malic acid gets accumulated and decreases pH. 59

60. Mechanism of CAM (Daytime) Malic acid gets converted into starch, glucose, etc. in the presence of light. Decarboxylation of malic acid during daytime yields CO2 inside the photosynthetic tissues and this CO2 is fixed by C3 cycle. Malic acid ----------------- (malic enzyme) Pyruvic acid + CO2. Thus, CO2 fixation takes place without CO2 entry directly from air. During daytime, the pH is comparatively higher than that of night. It undergoes decarboxylation to form pyruvic acid and CO2. This CO2 enters in C3 cycle and pyruvic acid goes for regeneration of PEP. In CAM plants however, both C3 and C4 pathways occur in mesophyll cells. C4 and C3 and pathways occur simultaneously in C4 plants while in CAM plants they occur during night and day respectively. Thus, C3 and C4 pathways are separated in space in C4 plants while in CAM plants C3 and C4 pathways are separated in time. 60

61. CAM pathway 61

62. Significance of CAM CAM cycle is considered as the environmental adaption because most of CAM plants grow in xeric conditions. It is an important physiological and biochemical adaption of photosynthetic carbon metabolism to water-stress. CAM plants have evolved mechanism for optimum utilization of CO2 with minimum loss of water. In CAM plants CO2 is stored in the form of malic acid and the decarboxylation of malic acid results in the release of CO2 during daytime. 62

63. Significance of CAM During day light, amount of titratable acidity decreases significantly (with increase of cell sap pH) known as light deacidification. During dark, increased amount of titratable acidity (with decrease of cell sap pH) known as dark acidification. Portulaca oleracea (C4 plant) may show CAM activity under certain environmental conditions. In Kalachoe blossfeldiana short days may induce CAM in young leaves than due to factor of ageing. 63

64. Photorespiration Decker and Tio (1959) coined the term photorespiration . It was generally believed up to mid-1950 that the rate of respiration of a green leaf is the same in light as well as in dark. But it was found that the rate of respiration in chlorophyllous tissues of higher plants (except many monocots) increases in the light than in dark. The phenomenon of increased rate of respiration induced by light is called photorespiration. It is nothing but the release of CO 2 in respiration in the presence of light. The respiratory substrate in normal respiration is glucose but for photorespiration it is glycolic acid (2C). It is found that under conditions of high O 2 concentration (high O 2 /low CO 2 ). Rubisco does not fix CO 2 but undergoes oxygenase activity. Site of photorespiration: Three organelles viz. chloroplast, peroxisome and mitochondria are involved in the process of photorespiration. 64

65. Evidence for occurrence of photorespiration Experimental evidences reported by RGS Bidwell and et al (1969) shown that the light stimulated release of CO 2 (called photorespiration) contains a high proportion of carbon fixed. He feed tobacco leaves with 14 C radiotracers and revealed that first carbon atom of glycolic acid is liberated as CO 2 .The liberation of CO 2 is related to rise in temperature from 25 o C to 35 o C during photorespiration. The carboxylic group of glycolate is thought to be donor of CO 2 to photorespiratory release. Zelitch (1966) also proved the presence of photorespiration by using glycolate oxidase inhibitor. The work of Tregunna (1966) and Hew (1968) shown that the effects of light on CO 2 production are eliminated by loss of chlorophylls from leaves (due to mutation or nutrient deficiency). In non- chlorophyllous cells, the photorespiration process is absent. Illuminated corn (maize) leaves which do not release CO 2 in the CO 2 free air begin to release when treated with photosynthetic inhibitors like DCMU (Dichlorophenyl-1-Dimethyl Urea). This inhibitor does not check the dark respiration. 65

66. Normal respiration and photorespiration 66   Feature Normal respiration Photorespiration 1 Respiratory substrate Carbohydrate or fat or protein Glycolic acid (2C) 2 Occurrence In all living cells In photosynthetic cells 3 Biosynthesis of substrate Substrate may be recently formed or stored Substrate always recently formed 4 Site Cytoplasm and mitochondria Chloroplasts 5 H 2 O 2 Not formed Formed 6 ATP Several are formed No ATP formed. 7 NAD and NADH NAD reduced to NADH NADH oxidized to NAD 8 Transamination Does not occur It occurs 9 O 2 concentration Not dependent totally on O 2 conc. Depend totally on O 2 conc.

67. Steps of Photorespiration 1. Oxygenase activity: RuDP ------ ( Rubisco O 2 ) Phosphoglylic acid (2C)+ 3 PGA(3C). 2. Phosphoglycolic acid undergoes dephosphorylation to form glycolic acid . Phosphoglycolic acid + H 2 O -----( phosphatase ) Glycolic acid + Phosphoric acid. 3. In peroxisomes glycolic acid is converted into glyoxylic acid in presence of glycolate oxidase. Glycolic acid ---------( glycolate oxidase ) glycolic acid. 4. Formation of H 2 O 2 : Glyoxylic acid+O 2 ----( glycolate oxidase , light) glyoxylic acid + H 2 O 2 . 5. H 2 O 2 is converted into O 2 and water in presence of catalase . 2H 2 O 2 ----------( catalase ) 2 H 2 O+O 2 . 6.Glyoxylic acid+glutamic acid ----( aminotransferase ) glycine +  -ketoglutarate. Glyoxylic acid is converted into glycine in presence of glutamate glyoxylate aminotransferases. Glycine is then transported to mitochondria via cytosol. 67

68. Steps of Photorespiration 7. In mitochondria two glycine molecules reach to produce serine , CO 2 and NH 3 . 2 Glycine + H 2 O + NAD + ----------- ( multienzyme ) Serine + CO 2 + NH 3 + NADH. At this place light induced CO 2 is liberated. Ammonia released by mitochondria is assimilated by chloroplast within the same cell. 8. Serine is then transported out of mitochondria into peroxisome where it is converted into hydroxy pyruvic acid and glyceric   acid. Serine ------------ hydroxy pyruvic acid + glyceric acid. 9. Finally glyceric acid is transported to chloroplast where it undergoes phosphorylation by ATP to form 3-PGA which is ultimately used in photosynthetic carbon reduction cycle. Thus, overall process becomes cyclic series of reactions and may be called photorespiratory carbon oxidation   68

69. Photorespiration 69

70. Significance of Photorespiration The photorespiration occurs in the temperate plants while tropical plants do not show this type of mechanism. Presence of photorespiration process decreases the photosynthetic efficiency of plants. The presence of photorespiration process decreases the photosynthetic potential (under certain circumstances as much as 50%). Up till now what is the actual role of photorespiration is not known. According to one hypothesis photorespiration provides the protective mechanism against the light destruction of chloroplasts in C 3 plants. This mechanism reduces the oxygen injury to chloroplast. By consuming oxygen, the process helps in maintaining low oxidative state in chloroplast of C 3 plants. According to some research workers photorespiration utilizes the excess of ATP and reducing power (NADPH) produced at higher levels of light. 70

71. Significance of Photorespiration At least it appears that photorespiration has the following functions in plants. i ) Amino acids like glycine and serine are synthesized during photorespiratory metabolism. ii) These are the precursors for many important metabolites such as proteins, chlorophyll, nucleotides, etc. iii) Conversion of glycolic acid to glyoxylate consumes NADH (which is generated in the light reaction of photosynthesis). Thus, photorespiration involved in dissipating excess reducing power. Glycolic acid is formed during this process may be involved the protection against the destruction action of photo-oxidation. Many scientists have questioned the significance of these functions. According to some scientists, RuBP carboxylase apparently emerged early in the evolution when atmosphere was rich in CO 2 and almost devoid of O 2 . However, as the CO 2 content of air increased it started functioning as oxygenase as well. Thus, it is inevitable process as it consequence of CO 2 / O 2 concentration. 71

72. Chemiosmotic theory The word chemiosmosis refers to conversion of chemical energy e.g., in the oxidation of NADH by oxygen) to osmotic energy (i.e. difference in the concentration of proton on two sides of mitochondrial membrane) the membrane. Energy released by proton flow is used to form ATP from ADP and Pi. This hypothesis provides general mechanism for coupling the energy in electron transport to phosphorylation. This hypothesis was proposed by Peter Mitchell in (1961). This was proposed to explain the process of ATP formation both in respiration and photosynthesis (photophosphorylation). 72

73. Chemiosmotic theory This hypothesis having five main postulates. The reactions occur on thylakoid membrane which forms closed vesicles and which is almost impermeable to the passive flow of protons. Electron donors and acceptors are arranged vectorially in the membrane. Electron transfer is obligatory to the pumping of hydrogen ions from the stroma into the osmotic space of the intrathylakoid membrane, leading to build-up of a transmembrane proton concentration (  PH) and the transmembrane field (  ) The combination of (  PH) and (  ) for as store of energy, tending to expel protons from the intrathylakoid space. There is a back flow of protons via the enzyme ATP synthetase (also called Coupling factor) located across Support to this theory: Data from several types of experiments support this hypothesis. 73

74. ATP synthesis through chemiosmosis in non-cyclic photophosphorylation 74

75. Significance of Chemiosmotic theory It provides understanding about biological energy transductions including process of oxidative phosphorylation in mitochondria and phosphorylation in chloroplasts. The mechanism of energy coupling is similar in both cases. The conservation of free energy involves the passage of electrons through a chain to membrane bound oxidation-reduction (redox) carriers and concomitant pumping of protons across the membrane producing electrochemical gradient the proton motive force. The force drives the synthesis of ATP by membrane bound enzyme complexes through which protons flow back across the membrane, down their electrochemical gradient proton motive force also drives other energy requiring processes of cells. The chemiosmotic theory also explains the formation of ATP in thylakoid membrane of chloroplast. In these cases, the transfer of protons across the thylakoid membrane occurs through alternate reduction (oxidation) of plastoquinone. This plastiquinone plays similar role as that of ubiquinone plays in ETS chain of mitochondria. In photosynthesis the energy required for the flow of electrons from water to NADP + and for transport of protons across the membrane is supplied by absorption of light. 75

76. Questions on Photosynthesis What are C4 plants? Describe the biochemical reactions involved in HSK pathway. What is photorespiration? Give schematic representation of glycolate cycle. Describe Crassulacean Acid Metabolism and give its significance. What is photophosphorylation? Give an account of chemiosmotic mechanism of photophosphorylation. What is oxidative phosphorylation? Explain ATP synthesis by chemiosmotic theory. Write about Red drop and Emmerson effect. Describe the mechanism of cyclic and non-cyclic photophosphorylation. What are C 3 plants? Describe the various steps involved in C 3 cycle. Describe the role of pigments in photosynthesis. Describe HSK pathway. 76

77. Questions on Photosynthesis 11. Explain cyclic photophosphorylation. 12. ‘Photosynthesis to certain extent is reverse of respiration’ Justify and amplify the statement. 13. What are photosystems? Explain the working of both the photosystems in photosynthesis. 14. Compare C3 and C4 plants. 15. Give the composition and function of two photosystems. 16. Write a note on the Hill reaction. 17. Write an account on CAM pathway. 18. Write short note on i ) Quantasomes ii) Emmerson effect 77