Photosynthesis in Higher Plants - PDF

120 C 03 Photosynthesis in higher plants h apter ontents 56. Introduction 181 57. What do we know...

120 C 03 Photosynthesis in higher plants h apter ontents 56. Introduction 181 57. What do we know 181 58. Early experiments 182 59. Where does photosynthesis take place 183 60. How many pigments are involved in photosynthesis 183 61. Mechanism of photosynthesis 186 62. What is light reaction 186 63. Chemiosmotic hypothesis 189 64. Where are the ATP and NADPH used? 191 (Dark reaction/Biosynthestic phase) 65. C3 pathway or the Calvin cycle 192 66. C4 pathway/CO2 concentrating mechanism/ Co-operative photosynthesis/Dicarboxylic acid cycle (DCA cycle)/Hatch and Slack pathway 193 67. Photosynthetic carbon oxidation cycle (PCO)/C2 cycle/photorespiration/glycolate-metabolism 195 68. Factors affecting photosysnthesis 197 69. Exercise-I (Conceptual Questions) 200 70. Exercise-II (Previous Years Questions) 203 71. Exercise-III (Analytical Questions) 205 NEET SYLLABUS Photosynthesis : Photosynthesis as a means of Autotrophic nutrition; Site of photosynthesis take place; pigments involved in Photosynthesis (Elementary idea); Photochemical and biosynthetic phases of photosynthesis; Cyclic and non cyclic and photophosphorylation; Chemiosmotic hypothesis; Photorespiration C3 and C4 pathways; Factors affecting photosynthesis.  123 MELVIN CALVIN born in Minnesota in April, 1911 received his Ph.D. in Chemistry from the University of Minnesota. He served as Professor of Chemistry at the University of California, Berkeley. Just after world war II, when the world was under shock after the Hiroshima-Nagasaki bombings, and seeing the illeffects of radio- activity, Calvin and co-workers put radioactivity to beneficial use. He along with J.A. Bassham studied reactions in green plants forming sugar and other substances from raw materials like carbon dioxide, water and minerals by labelling the carbon dioxide with C14. Calvin proposed that plants change light energy to chemical energy by transferring an electron in an organised array of pigment molecules and other substances. The mapping of the pathway of carbon assimilation in photosynthesis earned him Nobel Prize in 1961. The principles of photosynthesis as established by Calvin are, at present, being used in studies on renewable resource for energy and materials and basic studies in solar energy research.  ALLEN Pre-Medical : Biology 181 PHOTOSYNTHESIS IN HIGHER PLANTS INTRODUCTION * "Photosynthesis is a Physico - chemical or photo–biochemical process in which the organic compounds (carbohydrates) are synthesised from the inorganic raw material (H2O & CO2) in the presence of light & pigments. O2 is evolved as by product or one of the net products". Sunlight 6CO2 + 12H2O C6H12O6 + 6H2O + 6O2 Chlorophyll * Light energy is converted into chemical energy by photosynthesis. * Photosynthesis is a redox reaction during which oxidation of H2O occurs (as it provides H+ and e–) during light reaction and reduction of CO2 occurs (as it accepts H+ & e–) during dark reaction (biosynthetic phase) * Photosynthesis is an Anabolic (synthesising) & Endergonic (Energy absorbing) process. WHAT DO WE KNOW * Some simple experiments show that chlorophyll (green pigment of the leaf), light and CO2 are required for photosynthesis to occur. * Look for starch formation in two leaves – a variegated leaf or a leaf that was partially covered with black paper and one that was exposed to light. On testing these leaves for starch it was clear that photosynthesis occurred only in the green parts of the leaves in the presence of light. * Another experiment is the Moll's half-leaf experiment, where a part of a leaf is enclosed in a test tube containing some KOH soaked cotton (which absorbs CO2), while the other half is exposed to air. The setup is then placed in light for some time. On testing for starch later in the two halves of the leaf, the exposed part of the leaf tested positive for starch while the portion that was in the tube, tested negative. This shows that CO2 is required for photosynthesis. Z:\NODE02\B0B0-BA\NURTURE\BIO\ENG\MODULE_3\03-PLANT PHYSIOLOGY\04-PHOTO-E.P65 Leaf attached Area of leaf with plant unaffected with lodine Black paper Starch test strip Areas of leaf Clip to hold affected with paper Iodine Moll's Half leaf experiment Variegated leaf experiment E 182 Pre-Medical : Biology ALLEN EARLY EXPERIMENTS J. Priestley :– * Joseph Priestley (1733-1804) in 1770 performed a series of experiments that revealed the essential role of air in the growth of green plants. * Priestley observed that a candle burning in a closed space – a bell jar, soon gets extinguished. Similarly, a mouse would soon suffocate in a closed space. He concluded that a burning candle or an animal that (a) (b) breathe the air, both somehow, damage the air. But when he placed a mint plant in the same bell jar, he found that the mouse stayed alive and the candle continued to burn. q Prisetley hypothesised as follows : plants restore to the air whatever breathing animals and burning candles remove. (c) (d) Priestley’s experiment Jan Ingenhousz (1779) :– Using a similar setup as the one used by Priestley, but by placing it once in the dark and once in the sunlight, Jan Ingenhousz (1730 - 1799) showed that sunlight is essential to the plant process that somehow purifies the air fouled by burning candles or breathing animals. Ingenhousz in an elegant experiment with an aquatic plant showed that in bright sunlight, small bubbles were formed around the green parts while in the dark they did not. Later he identified these bubbles to be of oxygen. Hence he showed that it is only the green part of the plants that could release oxygen. J. V. Sachs (1854) :– It was not until about 1854 that Julius von Sachs provided evidence for production of glucose when plants grow. Z:\NODE02\B0B0-BA\NURTURE\BIO\ENG\MODULE_3\03-PLANT PHYSIOLOGY\04-PHOTO-E.P65 Glucose is usually stored as starch. His later studies showed that the green substance in plants (chlorophyll as we know it now) is located in special bodies (later called chloroplasts) within plant cells. He found that the green parts in plants is where glucose is made, and that the glucose is usually stored as starch. Van Neil (1897-1985) :– A milestone contribution to the understanding of photosynthesis was that made by a microbiologist, Cornelius van Niel, who, based on his studies of purple and green bacteria, demonstrated that photosynthesis is essentially a light-dependent reaction in which hydrogen from a suitable oxidisable compound reduces carbon dioxide to carbohydrates. Light 2H2 A + CO2 ¾¾¾ ® 2A + CH2O + H2O H2A = suitable oxidisable compound or H-Donor In green plants H2O is the hydrogen donor and is oxidised to O2. Some organisms do not release O 2 during photosynthesis. When H2S, instead is the hydrogen donor for purple and green sulphur bacteria, the ‘oxidation’ product is sulphur or sulphate depending on the organism and not O2. Hence, he inferred that the O2 evolved by the green plant comes from H 2O, not from carbon dioxide. E  ALLEN Pre-Medical : Biology 183 Ruben & Kamen (1941) :– Used O18 (radioisotopic technique) to show experimentally that O2 in photosynthesis released from water. Light 6CO2 + 12H2O18 ¾ ¾¾ ¾® C6H12O6 + 6H2O + 6O218 Chlorophyll WHERE DOES PHOTOSYNTHESIS TAKE PLACE * Photosynthesis does take place in the green leaves of plants but it does so also in other green parts of the plants. * Within the chloroplast there is the membranous system consisting of grana, the stroma lamellae, and the matrix stroma. There is a clear division of labour within the chloroplast. The membrane system is responsible for trapping the light energy and also for the synthesis of ATP and NADPH. In stroma, enzymatic reactions synthesise sugar, which in turn forms starch. The former set of reactions, since they are directly light driven are called light reactions (photochemical reactions). The latter are not directly light driven but are dependent on the products of light reactions (ATP and NADPH). Hence, to distinguish the latter they are called, by convention, as dark reactions (carbon reactions). SUN H 2O CO 2 Light STROMA + NADP ADP + Pi Light reactions Dark reactions (Granum) for reduction for oxidation of CO 2 of H 2O ATP – e + NADPH.H GRANUM H+ Z:\NODE02\B0B0-BA\NURTURE\BIO\ENG\MODULE_3\03-PLANT PHYSIOLOGY\04-PHOTO-E.P65 CHLOROPLAST O2 Sugar Starch & other organic compounds Division of labour in chloroplast HOW MANY PIGMENTS ARE INVOLVED IN PHOTOSYNTHESIS * Photosynthetic pigments are special molecules those absorb, transmitt and reflect different colours of light from the visible spectrum of sunlight. Pigment appears in the colour which it reflect and uses the colour which it absorbs. PAR (Photosynthetic Active radiation) – 400–700 nm * Photosynthetic Pigments are of following types :– 1. Chlorophylls 2. Carotenoids 3. Phycobilins E 184 Pre-Medical : Biology ALLEN 1. Chlorophylls – Green colored pigment Light is required for their synthesis Soluble in organic solvents * Chlorophylls are of following types – Chl. – a, Chl – b, Chl. – c, Chl. – d, Chl. – e * Chl – a is universal pigment, which is found in all O 2 liberating photosynthetic organisms. Its color is blue green in chromatogram. * Chl – b is accessory photosynthetic pigment found in euglenoids, green algae and higher plants. Its color is yellowish green in chromatogram * Chlorophyll – a C55H72O5N4Mg -® CH3 group in IInd pyrrol ring. * Chlorophyll – b C55H70O6N4Mg -® CHO group in IInd pyrrol ring. q Structure of chlorophyll – H CH2 C H CH3 CHO H3C I II C2H5 II C2H5 N N H Mg H Chlorophyll (b) N N H3C IV III CH3 H CH2 H H CH2 COOCH3 O Chlorophyll (a) HC – CH3 HC – CH3 C – CH3 CH3 CH3 (CH 2)3 (CH2)3 (CH2)3 CH2 CH CH O O=C Structure of chl-a look like tadpole. Z:\NODE02\B0B0-BA\NURTURE\BIO\ENG\MODULE_3\03-PLANT PHYSIOLOGY\04-PHOTO-E.P65 Chlorophyll Porphyrin Head Phytol Tail (C20H39OH) l Size = 15 × 15 Å l Size = 20 Å long l It is hydrophilic l It is hydrophobic l Its a tetrapyrole structure l Tail remain embedded in with Mg present in the centre lipid bilayer of thylakoid membrane. q Chlorophyll synthesis – * light Succinyl CoA + Glycine -® Protochlorophyll ¾¾¾ 2H ® Chlorophyll. This reaction is catalysed by iron (Fe) E  ALLEN Pre-Medical : Biology 185 2. Carotenoids (Yellow to yellow orange) – * The first carotenoid was discovered in carrot and was named carotene. Carotenes are the first type of Carotenoids. These are yellow orange in colour. It contains carbon and hydrogen. * Another carotenoid is xanthophyll that contains carbon, hydrogen and oxygen. These are usually yellow in colour. * Among carotenoids, b – carotene (type of carotene) and lutein (type of xanthophyll) are common in plants. 3. Phycobillins : · They are hot water soluble pigment. · They lack Mg and phytol tail. Types : (i) Phycocyanin – Blue (ii) Phycoerythrin – Red (iii) Allophycocyanin – Light blue They occur exclusively in BGA and Red algae as an accessory pigments. Absorption spectrum * Graphical presentation of the Chlorophyll b Absorbance of light by chloroplast pigments absorption of different wavelenght of Carotenoids light by a particular pigment. Chlorophyll a * Graph (a) showing the ability of pigments to absorb lights of different wavelengths (absorption spectrum). Chl. – a shows maximum (a) absorption at blue light. It show another absorption peak at red light. (measured by O 2 release) Rate of photosynthesis Action spectrum * The graphic curve depecting the relative rates of photosynthesis at different wavelengths of light is called action spectrum (b) * Figure (b) showing the wavelengths at which Rate of photosynthesis photosynthesis occurs in a plant (Action Z:\NODE02\B0B0-BA\NURTURE\BIO\ENG\MODULE_3\03-PLANT PHYSIOLOGY\04-PHOTO-E.P65 Absorption spectrum). Light absorpbed Figure (c) show that the wavelengths at which there is maximum absorption by chlorophyll a, i.e., in the blue and the red regions, also show higher rate of photosynthesis. Hence, we can conclude that chlorophyll –a is the (c) 400 500 600 700 chief pigment associated with Wavelength of light in nanometres (nm) photosynthesis. E 186 Pre-Medical : Biology ALLEN Wavelength of light (nm) Now consider the interesting experiment 400 500 700 600 done by T.W Engelmann. Using a prism he Aerobic bacteria split light into its spectral components and Cladophora then illuminated a green alga, Cladophora, Chloroplast placed in a suspension of aerobic bacteria. The bacteria were used to detect the sites of O2 evolution. He observed that the bacteria accumulated mainly in the region of blue and red light of the split spectrum. A first action spectrum of photosynthesis was thus described. It resembles roughly the absorption spectra of chlorophyll a and b. Prism White Light * These graphs a, b and c together show that most of the photosynthesis takes place in the blue and red regions of the spectrum; some photosynthesis does take place at the other wavelengths of the visible spectrum. It happens because other thylakoid pigments like chlorophyll b, xanthophylls and carotenoids, which are called accessory pigments, also absorb light and transfer the energy to chlorophyll a. Indeed, they not only enable a wider range of wavelength of incoming light to be utilised for photosynthesis but also protect chlorophyll a from photo-oxidation. * It resembles roughly the absorption spectra of chlorophyll a and b. MECHANISM OF PHOTOSYNTHESIS There are two types of reactions in photosynthesis – 1. Light reaction 2. Dark reaction Z:\NODE02\B0B0-BA\NURTURE\BIO\ENG\MODULE_3\03-PLANT PHYSIOLOGY\04-PHOTO-E.P65 WHAT IS LIGHT REACTION ? * Light reactions or the 'Photochemical' phase include light absorption, water splitting, oxygen release and formation of high energy chemical intermediates like ATP and NADPH. * Photosystems are required for this process. * Emerson & Arnold worked on chlorella and gave concept of two photosystem or two pigment system. * The groups of photosynthetic pigments in thylakoid membranes are known as photosystems. * The pigments are organised as two discrete photochemical light harvesting complexes (LHC) within the Photosystem I (PS I) and Photosystem II (PS II). Photosystem = Reaction centre + LHC E  ALLEN Pre-Medical : Biology 187 * In every photosystem there is a reaction centre (molecule of chlorophyll –a) surrounded by Primary acceptor accessory pigments. The accessory pigments absorb light energy and transfer it to the reaction centre. Th ese pigment s he lp t o make Reaction centre photosynthesis more efficient by absorbing Photon different wavelengths of light. These molecules are known as antenna molecules or LHC. The LHC Accessory Pigment molecules are made up of hundreds (250 - 400 molecules) (Antenna or LHC) of pigment molecules bound to proteins. * The reaction centre is different in both the Photosystem photosystems. In PS I the reaction centre chlorophyll –a has an absorption peak at 700 nm, hence is called P700, while in PS II it has absorption peak at 680 nm, and is called P680. * These are named in the sequence of their discovery, and not in the sequence in which they function during the light reaction. * The PS II is located in the appressed region of granal thylakoids and PS I in non appressed region of grana and in stroma thylakoids. (In the other way we can say that granal thylakoids have both PS I and PS II whereas stroma thylakoids have only PS I). PHOTOPHOSPHORYLATION Z:\NODE02\B0B0-BA\NURTURE\BIO\ENG\MODULE_3\03-PLANT PHYSIOLOGY\04-PHOTO-E.P65 * Synthesis of ATP from ADP and inorganic phosphate (iP) with the help of light energy is known as photophosphorylation. It is of two types :- (I) Non-cyclic Photophosphorylation / Z-Scheme - * Both PS–I and PS–II are involved in Non cyclic photophosphorylation. So It occurs at grana thylakoids only, not in stroma thylakoids because stroma thylakoids lack PS - II as well as NADP reductase enzyme. * Primary e– acceptor from PS-II is pheophytin, which passes electrons to an electrons transport system (ETS) consisting of cytochromes. This movement of electrons is downhill, in terms of an oxidation- reduction or redox potential. The electrons emitted from PS II pass through the electron transport chain to the pigments of PS I. Simultaneously, electrons in the reaction centre of PS I are also excited when they receive red light of wavelength 700 nm and are transferred to another accepter molecule (FRS) and then to Fd. These electrons then move downhill again, this time to a molecule of energy rich NADP+. E 188 Pre-Medical : Biology ALLEN Photosystem II Photosystem I e– acceptor NADPH Light e acceptor – + ADP + iP ATP NADP Electron transport system LHC LHC – + H 2O ® 2e + 2H + [O] + 2H t 2e – gh PQ Li Pheo PQH2 – C PQH2 2e Cytb PS-II OE 6 2H + 1 O2 & + – 2e 2 C yt f + 2H 2e– Li H2O gh PC t – Thylakoid + + H H H Proton + 2e F membrane H+ H+H+H+ gradient RS PS-I + + Lumen HH Fd + + 2e – NADP + 2H NADP reductase + Stroma NADPH + H 3H + Chemiosmosis Z:\NODE02\B0B0-BA\NURTURE\BIO\ENG\MODULE_3\03-PLANT PHYSIOLOGY\04-PHOTO-E.P65 ADP+Pi ATP The electrons that were moved from photosystem II must be replaced. This is achieved by electrons available due to splitting of water. The splitting of water is associated with the PS II; water is split into H+, [O] and electrons. This creates oxygen, one of the net products of photosynthesis. The electrons needed to replace those removed from photosystem I are provided by photosystem II. 2H2O ¾® 4H+ + O2 + 4e– We need to emphasise here that the water splitting complex is associated with the PS II, which itself is physically located on the inner side of the membrane of the thylakoid. * The addition of these electrons reduces NADP+ to NADPH + H+ (Protons from stroma). At every step oxidation/ reduction of e– carrier take place and energy is released which is utilized in creation of proton gradient to form ATP. E  ALLEN Pre-Medical : Biology 189 * This whole scheme of transfer of electrons, starting from the PS II, uphill to the acceptor, down the electron tranport chain to PS I, excitation of electrons, transfer to another accepter, and finally down hill to NADP+ is called the Z scheme, due to its characteristic shape. * This Z shape is formed when all the carriers are placed in a sequence on a redox potential scale. (II) Cyclic Photophosphorylation- * In cyclic photophosphorylation, only PS–I (LHC–I) works. * A possible location where this could be happening is in the stroma lamellae/thylakoid * During Cyclic ETS, the electron ejected from reaction centre of PS-I, returns back to its reaction centre. At every step oxidation and reduction of each e– carrier take place which release the energy and this energy is utilized in creation of proton gradient to form ATP. * In cyclic ETS, no oxygen evolution occurs because photolysis of water is absent. * NADPH + H+ (Reducing power) is not formed in cyclic process. There is formation of only ATP. Photosystem I * – 2e P700 FRS – 2e– e acceptor 2e– Fd Light 2e– TP iP A (Stroma) PQ Cyt–b6 2H+ & P+ Electron 2e– Cyt–f AD transport PQH2 h t system 2e– Lig + PC 2H (Lumen) 2e– PS-I Chlorophyll P700 P 700 CYCLIC PHOTOPHOSPHORYLATION Non-cyclic photophosphorylation Cyclic photophosphorylation (1) Both PS–II & PS–I are involved in non– (1) Only PS–I is involved in cyclic process. Z:\NODE02\B0B0-BA\NURTURE\BIO\ENG\MODULE_3\03-PLANT PHYSIOLOGY\04-PHOTO-E.P65 cyclic process. (2) First e– acceptor from PS–II is pheophytin (2) First e– acceptor from PS–I is FRS (3) The e– expelled from reaction center (3) The e– expelled from P700 is is not cycled back. Its loss is cycled back. – compensated by e from H2O. (4) Photolysis of water and evolution of (4) Photolysis of water and evolution O2 takes place. of O2 does not take place. (5) NADP is reduced to NADPH + H. + + (5) NADP+ is not reduced. q Chemiosmotic Hypothesis – * Proposed by Peter Mitchell (1961) to explain the mechanism of ATP formation (Phosphorylation) in chloroplast (During photosynthesis) and in mitochondria (During respiration). * According to this hypothesis ATP synthesis is linked to developement of a proton gradient (Difference of proton (H+) concentration) across a membrane. In chloroplast these are membranes of the thylakoid. E 190 Pre-Medical : Biology ALLEN * The steps that cause a proton gradient to develop – (a) Since splitting of the water molecule takes place on the inner side of the membrane, the hydrogen ions that are produced by the splitting of water accumulate within the lumen of the thylakoids. (b) As electron move through the photosystems, protons are transported across the membrane. This happens because the primary accepter of electron (Pheophytin) which is located towards the outer side of the membrane transfers its electron not to an electron carrier but to an H carrier (Plastoquinone). Hence this molecule removes a proton from the stroma while transporting an electron. When Plastoquinone passes on its electron to the electron carrier (FeS protein 'or' Cytochrome) on the inner side of the membrane, the proton is released into the inner side of the membrane or the lumen side of the membrane. (c) The NADP reductase enzyme is located on the stroma side of the membrane. Along with electrons that come from the accepter of electrons (Fd) of PS I, protons are necessary for the reduction of NADP+ to NADPH+ + H+. These protons are also removed from the stroma. + NADP + H + NADPH + Stroma (low H ) H + Light FNR Light Cytochrome Fd B6f P700 P680 PQ PQH 2 PS-I PS-II PC inone Plastoqu H + Plastocyanin H2O ½ O2 + H + Oxidation of water H + + H+ H High Lumen Electrochemical H + + (high H ) Potential CF0 Thylakoid Gradient membrane Stroma Low ATP Z:\NODE02\B0B0-BA\NURTURE\BIO\ENG\MODULE_3\03-PLANT PHYSIOLOGY\04-PHOTO-E.P65 synthase CF1 FNR (Ferradoxine NADP reductase) ADP + Pi H ATP + ATP synthesis through chemiosmosis * Hence, within the chloroplast, protons in the stroma decrease in number, while in the lumen there is accumulation of protons. This creates a proton gradient across the thylakoid membrane as well as a measurable decrease in pH in the lumen. * The ATPase enzyme consists of two parts: one called the F0 is embedded in the membrane and forms a transmembrane channel that carries out facilitated diffusion of protons across the membrane. The other portion is called F1 and protrudes on the outer surface of the thylakoid membrane on the side that faces the stroma. * This gradient is important because it is breakdown of this gradient that leads to release of energy. The gradient is broken down due to the movement of protons across the membrane to the stroma through the transmembrane channel of the F0 of the ATPase. E  ALLEN Pre-Medical : Biology 191 * The break down of the gradient provides enough energy to cause a conformational change in the F1 particle of the ATPase, which makes the enzyme synthesise several molecules of energy-packed ATP." * "Chemiosmosis requires a membrane, a proton pump, a proton gradient and ATPase. Energy is used to pump protons across a membrane, to create a gradient or a high concentration of protons within the thylakoid lumen. ATPase has a channel that allows diffusion of protons back across the membrane; this releases enough energy to activate ATPase enzyme that catalyses the formation of ATP." 1. Who, after conducting experiments on purple and green sulphur bacteria, inferred that O2 evolved during photosynthesis comes from H2O not from CO2? (1) Sachs (2) Engelmann (3) Van Niel (4) Blackmann 2. Which of the following is/are formed during Z-scheme of photophosphorylation? (1) ATP (2) NADPH2 (3) O2 (4) All of these 3. In cyclic photophosphorylation, the electron released by reaction centre (P700) is ultimately accepted by :- (1) Ferredoxin (2) NADP+ (3) Reaction centre (P700) (4) Plastocyanin 4. During cyclic photophosphorylation, the biochemical objective of PS I is to :- (1) Oxidise NADPH (2) Hydrolyse ATP (3) Phosphorylate ADP (4) Reduce NADP+ 5. During the process of photosynthesis the raw materials used are :- (1) Glucose (2) Chlorophyll (3) Starch (4) CO2 and H2O WHERE ARE THE ATP AND NADPH USED? (Dark Reaction/Biosynthetic phase) (i) In this process CO2 is reduced to sugar. (ii) It is known as dark reaction but it doesn't mean that it occurs in dark. (iii) This process does not directly depend on the presence of light but is dependent on the products of the light reaction like ATP and NADPH2 so immediately after light becomes unavailable, the biosynthetic process continues for some time and then stops. If then light is made available, the synthesis starts again. * It was of interest to scientists to find out how this reaction proceeded, or rather what was the first product formed when CO2 is taken into a reaction or fixed. Just after world war II, among the several efforts to put radioisotopes Z:\NODE02\B0B0-BA\NURTURE\BIO\ENG\MODULE_3\03-PLANT PHYSIOLOGY\04-PHOTO-E.P65 to beneficial use, the work of Melvin Calvin is exemplary. The use of radioactive 14C by him in algal photosynthesis studies led to the discovery that the first CO 2 fixation product was a 3-carbon organic acid. He also contributed to working out the complete biosynthetic pathway; hence it was called Calvin cycle after him. The first product identified was 3-phosphoglyceric acid or in short PGA. * Scientists also tried to know whether all plants have PGA as the first product of CO 2 fixation, or whether any other product was formed in other plants. Experiments conducted over a wide range of plants led to the discovery of another group of plants, where the first stable product of CO 2 fixation was again an organic acid, but one which had 4 carbon atoms in it. This acid was identified to be oxaloacetic acid or OAA. Since then CO2 assimilation during photosynthesis was said to be of two main types: those plants in which the first product of CO2 fixation is a C3 acid (PGA), i.e., the C3 pathway, and those in which the first product was a C4 acid (OAA), i.e., the C4 pathway. * In different plants biosynthetic phase takes place by two different pathways 1. C3 pathway 2. C4 pathway E 192 Pre-Medical : Biology ALLEN * The CO2 reduction or assimilation into glucose in all photosynthetic plants take place by Calvin cycle, as it is the ultimate pathway of glucose synthesis operated in C3 and C4Plants. * In C3 pathway, biosynthetic phase has only Calvin cycle. In C4 pathway, some additional reactions also occur before Calvin cycle, during biosynthetic phase. 1. C3–pathway or The Calvin cycle :- * In C3–plants, calvin cycle occurs in stroma of chloroplast of mesophyll cells. * Ist stable compound of Calvin cycle is 3carbon compound–PGA (Phosphoglyceric acid or phosphoglycerate) thus Calvin cycle is called C3–cycle. (First compound is unstable, 6C keto acid–carboxy ketoribitol bisphosphate). * Calvin studied the dark reaction in green algae Chlorella & Scenedesmus. During his experiment he used radioisotopy (C 14 radioisotope) and chromatography techniques for identification and separation of intermediates of C 3–cycle. * RuBisCO (Ribulose-1,5-bis-Phosphate carboxylase-oxygenase) is main enzyme in C3–cycle which is present in stroma. RuBisCO is the most abundant enzyme and protein on earth. * For ease of understanding, the Calvin cycle can be described under three stages: carboxylation, reduction and regeneration. 1. Carboxylation – Carboxylation is the fixation of CO2 into a stable organic intermediate. Carboxylation is the most crucial step of the Calvin cycle where CO2 is utilised for the carboxylation of RuBP. This reaction is catalysed by the enzyme RuBP carboxylase which results in the formation of two molecules of 3-PGA. Since this enzyme also has an oxygenation activity it would be more correct to call it RuBP carboxylase-oxygenase or RuBisCO. 2. Reduction – These are a series of reactions that lead to the formation of glucose. The steps involve utilisation of 2 molecules of ATP for phosphorylation and two of NADPH for reduction per CO2 molecule fixed. The fixation of six molecules of CO2 and 6 turns of the cycle are required for the formation of one molecule of glucose from the pathway. 3. Regeneration – Regeneration of the CO2 acceptor molecule RuBP is crucial if the cycle is to continue uninterrupted. The regeneration steps require one ATP for phosphorylation to form RuBP. * Hence for every CO2 molecule entering the Calvin cycle, 3 molecules of ATP and 2 of NADPH are required. It is probably to meet this difference in number of ATP and NADPH used in the dark reaction that the cyclic phosphorylation takes place. Atmosphere (RuBP) CO2 + H2O Ribulose-1,5-bisphosphate (5C) Z:\NODE02\B0B0-BA\NURTURE\BIO\ENG\MODULE_3\03-PLANT PHYSIOLOGY\04-PHOTO-E.P65 Carboxylation 1 ADP 2 × 3-phosphoglycerate (3-PGA) Regeneration 3 Calvin - Cycle (3C) + 2ATP + 2NADPH + 2H ATP 2 Reduction 2 × (3-PGAL) + Triose 2ADP + 2Pi + 2NADP phosphate (3C) Glucose Sucrose, Starch E  ALLEN Pre-Medical : Biology 193 * 6 turns of Calvin cycle are required for the formation of one glucose as 6 CO2 are required for the synthesis of one hexose. * 12 NADPH + H+ & 18 ATP are required as assimilatory power to produce one Glucose in dark reaction in C3 cycle. Calvin cycle In Out Six CO2 One glucose 18 ATP 18 ADP 12 NADPH 12 NADP 2. C4 pathway/CO2 concentrating mechanism/Co-operative photosynthesis/ Dicarboxylic acid cycle (DCA cycle)/Hatch & Slack Pathway * Hatch & Slack (1967), Studied it in detail in sugarcane and maize leaves and proposed a new pathway for dark reactions. * First stable product of this pathway is OAA, which is a 4C, DCA (Dicarboxylic Acid), thus Hatch & Slack pathway is also called C4 pathway or DCA cycle. * Most of the C4 plants are monocots (Tropical grasses), which belong to Gramineae & Cyperaceae families. C4 plants are adapted to hot and dry environment. * E.g. of C4 plants – Sugarcane, Maize, Sorghum. * Wheat, Rice and Barley (monocot) are C3 species. * Kranz (Wreath) Anatomy – Present in leaves of C4 plants. In these plants special green large cells are found around the vascular bundles in leaves, these are called bundle sheath cells, and the leaves which have such anatomy are said to have 'Kranz anatomy'. 'Kranz' means 'wreath' and is reflection of the arrangement of cells. * The bundle sheath cells may form several layers around the vascular bundles, they are characterised by - (i) having a large number of chloroplasts (ii) thick walls impervious to gaseous exchange Z:\NODE02\B0B0-BA\NURTURE\BIO\ENG\MODULE_3\03-PLANT PHYSIOLOGY\04-PHOTO-E.P65 (iii) no intercellular spaces * Dimorphic chloroplasts are present in leaf cells of C4 plants. Chloroplast of bundle sheath cells or Kranz cells are large and without grana (Agranal i.e. the thylakoids are present only as stroma lamellae). Mesophyll chloroplast are small and with grana (Granal chloroplast i.e. both grana ans stroma thylakoid are present). * First CO2 acceptor in C4 plants is PEP (Phosphoenol Pyruvate) (3C–compound) in mesophyll cells, while second CO2 acceptor is RuBP (5C–compound), in bundle sheath cells. * Initial fixation of CO2 in mesophyll cells is catalysed by PEPcase (PEP carboxylase), which results in the formation of OAA(4C). * Then reduction of OAA take place by NADPH 2 results in formation of malic Acid (4C) or transamination of OAA resulting in formation of Aspartic acid (4C). * The malic acid or Aspartic acid (4C) formed in mesophyll cells is transfered to bundle sheath cells. In bundle sheath cells the oxidative decarboxylation of malate takes place and CO2 is released along with pyuruvic Acid (3C). E 194 Pre-Medical : Biology ALLEN Diagrammatic representation of the Hatch and Slack Pathway Atmospheric CO2 Mesophyll Plasma membrane cell Cell wall – HCO3 Phosphoenolpyruvate (PEP) (3C) Fixation 2ATP OAA (C4 acid) Regeneration NADPH NADP Plasmodesmata Malic/Aspartic acid(C4 acid) Pyruvic acid (C3 acid) Transport Bundle sheath cell Transport Malic/Aspartic acid Fixation by (C4 acid) Calvin cycle NADPH CO2 Decarboxylation Pyruvic acid (C3 acid) NADP Z:\NODE02\B0B0-BA\NURTURE\BIO\ENG\MODULE_3\03-PLANT PHYSIOLOGY\04-PHOTO-E.P65 * Released CO2 in bundle sheath cells is accepted by RUBP, catalysed by RuBisCO. The C3 cycle/calvin cycle operates in bundle sheath cells with utilization of assimilatory power (18 ATP & 12 NADPH2 ) resulting in formation of glucose. * Pyruvic acid from bundle sheath cells return to mesophyll cells. It regenerate the PEP (primary CO2 acceptor) by utilization of 12 ATP, catalysed by enzyme PPDK (Pyruvate phosphate dikinase). So in C4 plants total 30 ATP and 12 NADPH2 are utilized for synthesis of one glucose. * C4 plants are special because : (i) They have a special type of leaf anatomy (Kranz anatomy) (ii) They tolerate higher temperature (iii) They show a response to high light intensities (iv) They lack a process called photorespiration so have greater productivity of biomass. E  ALLEN Pre-Medical : Biology 195 * In C4 plants photorespiration does not occur. This is because they have a mechanism that increases the concentration of CO2 at the RuBisCO enzyme site. This takes place when the C4 acid (malic or aspartic acid) from the mesophyll cell is broken down in the bundle sheath cells to release CO2 (CO2 pumping), this results in increasing the intracellular concentration of CO2. In turn, this ensures that the RuBisCO functions as a carboxylase minimising the oxygenase activity. * In addition, in C4 plants, site of O2 evolution (mesophyll cell) and site of RuBisCO activity (Bundle sheath cell) are different. * The evolution of the C4 photosynthetic system is probably one of the strategies for maximising the availability of CO2 while minimising water loss. C4 plants are twice as efficient as C3 plants in terms of fixing carbon (making sugar). However, a C4 plant loses only half as much water as a C3 plant for the same amount of CO 2 fixed. Photosynthetic carbon oxidation (PCO) cycle/C2 Cycle/Photorespiration/ Glycolate–Metabolism * The light dependent uptake of O2 & release of CO2 in green cells of C3 plants are called Photorespiration. This process create an important difference between C3 and C4 plant. * It occurs in chloroplast, peroxisome & mitochondria. * RuBisCO is characterised by the fact that its active site can bind to both CO 2 and O2 – hence the name. This binding is competitive. It is the relative concentration of O 2 and CO2 that determines which of the two will bind to the enzyme. (Usually RuBisCO has a much greater affinity for CO2 than for O2). * Conditions for photorespiration – High light intensity (High O2, Low CO2) and high temperature. * Internal CO2 concentration is always remain almost constant around RuBisCO, but internal O2 concentration is variable (due to variable rate of light reaction). When O2 concentration is higher than CO2 concentration then Z:\NODE02\B0B0-BA\NURTURE\BIO\ENG\MODULE_3\03-PLANT PHYSIOLOGY\04-PHOTO-E.P65 RuBisCO perform oxygenation of RuBP, which leads to phenomenon of photorespiration. Here the RuBP instead of being converted to 2 molecules of PGA, binds with O2 to form one molecule of phosphoglycerate (3C) and one molecule of phosphoglycolate (2C). * In the photorespiratory pathway there is neither synthesis of sugars, nor of ATP and NADPH2. Rather it results in the release of CO2 with the utilisation of ATP. The biological function of photorespiration is not known yet. E 196 Pre-Medical : Biology ALLEN Characteristics C3 Plants C4 Plants Choose from Cell type in which the Calvin Mesophyll Bundle sheath Mesophyll/Bundle sheath/both cycle takes place Cell type in which the initial Mesophyll Mesophyll Mesophyll/Bundle sheath /both carboxylation reaction occurs How many cell types does the One Two Two: Bundle sheath and leaf have that fix CO2. mesophyll One: Mesophyll Three: Bundle sheath, palisade, spongy mesophyll Which is the primary CO 2 acceptor RuBP PEP RuBP/PEP/PGA Number of carbons in the 5 3 5/4/3 primary CO2 acceptor Which is the primary CO2 PGA OAA PGA/OAA/RuBP/PEP fixation product No. of carbons in the primary 3 4 3/4/5 CO2 fixation product Does the plant have RuBisCO? Yes Yes Yes/No/Not always Does the plant have PEP Case? Yes Yes Yes/No/Not always Which cells in the plant have Mesophyll Bundle sheath Mesophyll/Bundle sheath/none Rubisco? CO2 fixation rate under high Medium High Low/ high/ medium light conditions Whether photorespiration is Negligible Negligible High/negligible/sometimes Z:\NODE02\B0B0-BA\NURTURE\BIO\ENG\MODULE_3\03-PLANT PHYSIOLOGY\04-PHOTO-E.P65 present at low light intensities Whether photorespiration is High Negligible High/negligible/sometimes present at high light intensities Whether photorespiration would be High Negligible High/negligible/sometimes present at low CO2 concentrations Whether photorespiration would be Negligible Negligible High/negligible/sometimes present at high CO2 concentrations Temperature optimum 20-25°C 30-40°C 30-40 C/20-25C/above 40 C Examples Wheat Maize Rice Sugarcane Sorghum. E  ALLEN Pre-Medical : Biology 197 FACTORS AFFECTING PHOTOSYNTHESIS * An understanding of the factors that affect photosynthesis is necessary. The rate of photosynthesis is very important in determining the yield of plants including crop plants. Photosynthesis is under the influence of several factors, both internal (plant) and external. The plant factors include the number, size, age and orientation of leaves, mesophyll cells and chloroplasts, internal CO2 concentration and the amount of chlorophyll. The plant or internal factors are dependent on the genetic predisposition and the growth of the plant. * The external factors would include the availability of sunlight, temperature, CO2 concentration and water. As a plant photosynthesises, all these factors will simultaneously affect its rate. Hence, though several factors interact and simultaneously affect photosynthesis or CO2 fixation, usually one factor is the major cause or is the one that limits the rate. Hence, at any point the rate will be determined by the factor available at sub- optimal levels. * When several factors affect any [bio] chemical process, Blackman’s (1905) Law of Limiting Factors comes into effect. This states the following: * If a chemical process is affected by more than one factor, then its rate will be determined by the factor which is nearest to its minimal value: it is the factor which directly affects the process if its quantity is changed. * For example, despite the presence of a green leaf and optimal light and CO2 conditions, the plant may not photosynthesise if the temperature is very low. This leaf, if given the optimal temperature, will start photosynthesising. * CO2 is limiting in clear sky but light becomes limiting in cloudy days and in dense forest or for plants growing in shade. q EXTERNAL FACTORS – (1) Light – * We need to distinguish between light quality, light intensity and the duration of exposure to light, while discussing light as a factor that affects photosynthesis. There is a linear relationship between incident light and CO2 fixation rates at low light intensities. At higher light intensities, gradually the rate does not show further increase as other factors become limiting. Z:\NODE02\B0B0-BA\NURTURE\BIO\ENG\MODULE_3\03-PLANT PHYSIOLOGY\04-PHOTO-E.P65 Rate of photosynthesis B C E A D Light intensity * Light saturation occurs at 10% of full sunlight. * Except for plants in shade or in dense forests, light is rarely a limiting factor in nature. Increase in incident light beyond a point causes the breakdown of chlorophyll and a decrease in photosynthesis. E 198 Pre-Medical : Biology ALLEN (2) Temperature – * The dark reactions being enzymatic are temperature controlled. Though the light reactions are also temperature sensitive they are affected to a much lesser extent. * The C4 plants respond to higher temperatures (30° – 40° C) and show higher rate of photosynthesis while C3 plants have a much lower temperature optimum (20° – 25° C). * The temperature optimum for photosynthesis of different plants also depends on the habitat that they are adapted to. Tropical plants have a higher temperature optimum than the plants adapted to temperate climates. (3) CO2 – * Carbon dioxide is the major limiting factor for photosynthesis. The concentration of CO2 is very low in the atmosphere (between 0.03 and 0.04 percent). Increase in concentration upto 0.05 percent can cause an increase in CO2 fixation rate, beyond this the levels can become damaging over longer periods. * The C3 and C4 plants respond differently to CO2 concentrations. At low light conditions neither group responds to high CO2 conditions. At high