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Photosynthesis in Higher Plants, , , , , , In photosynthesis process, ‘energy rich compounds like, carbohydrates are synthesized from simple inorganic compounds, like carbon dioxide and water in the presence of chlorophyll and, sunlight with liberation of oxygen’. The process of photosynthesis, can also be defined as "transformation of photonic energy (i.e,, light or radiant energy) into chemical energy’, , About 90% of total photosynthesis in world is done by algae, in oceans and in freshwater, More than 170 billion tonnes of dry, matter are produced annually by this process, Further CO, fixed, annually through photosynthesis is about 7.0 x 10"kg., Photosynthesis is a reductive, anabolic and endothermic reaction., Photosynthesis helps to maintain the equilibrium position of O,, and CO, in the atmosphere., , Historical background, , Before seventeenth century it was considered that plants take, their food from the soil, , G_ Van Helmont (1648) concluded that all food of the, plant is derived from water and not from soil., , G Stephen Hales (Father of Plant Physiology) (1727), reported that plants obtain a part of their nutrition from air and, light may also play a role in this process., , Joseph Priestley (1772) demonstrated that green plants, (mint plant) purify the foul air (ie, Phlogiston), produced by, burning of candle, and convert it into pure air (I, Dephlogiston)., , Q dan Ingen-Housz (1779) concluded by his experiment, that purification of ait was done by green parts of plant only and, that too in the presence of sunlight. Green leaves and stalks, liberate dephlogisticated air (Having O,) during sunlight and, phlogisticated air (Having CO;) during dark,, , Jean Senebier (1782) proved that plants absorb CO,, and release O, in the presence of light. He also showed that the, rate of O, evolution depends upon the rate of CO, consumption,, , 1 Nicolus de Saussure (1804) showed the importance of, water in the process of photosynthesis. He further showed that the, amount of CO, absorbed is equal to the amount of O; released., , Q Julius Robert Mayer (1845) proposed that light has, radiant energy and this radiant energy is converted to chemical, energy by plants, which serves to maintain life of the plants and, also animals., , .Liebig (1845) indicated that main source of carbon in, plants is CO,,, , 2 Bousinganlt (1860) reported that the volume of CO,, absorbed is equal to volume of O, evolved and that CO,, absorption and O, evolution get start immediately after the plant, ‘was exposed to sunlight., , Julius Von Sachs (1862) demonstrated that fist visible, product of photosynthesis is starch. He also showed that, chlorophyll is confined to the chloroplasts., , Q Melvin Calvin (1954) traced the path of carbon in, photosynthesis (Associated with dark reactions) and gave the C,, cycle (Now named Calvin cycle). He was awarded Nobel prize in, 1961 for the technique to trace metabolic pathway by using, radioactive isotope., , Huber, Michel and Deisenhofer (1985) crystallised the, photosynthetic reaction center from the purple photosynthetic, bacterium, Rhodopseudomonas viridis, They analysed its structure, by X-ray diffraction technique. In 1988 they were awarded Nobel, prize in chemistry for this work.

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Photosynthesis in Higher Plants 657, , , , , , Photosynthesis in higher plants, , Chloroplast (The site of photosynthesis) : Chloroplast, are green plastids which function as the site of photosynthesis in, eukaryotic photoautotrops. It fixes CO, into carbohydrate., , Photosynthetic unit can be defined as number of pigment, molecules required to affect a photochemical act, that is the release, of a molecule of oxygen. Park and Biggins (1964) gave the term, quantasome for photosynthetic units which is equivalent to 230, chlorophyll molecules,, , Chloroplast pigments : Pigments are the organic molecules, that absorb light of specific wavelengths in the visible region due to, Presence of conjugated double bonds in their structures. The, chloroplast pigments are fat soluble and are located in the lipid part, of the thylakoid membranes (fret membrane). There is a wide, range of chloroplastic pigments which constitute more than 5% of, the total dry weight of the chloroplast. They are grouped under two, main categories :, , (2) Chlorophylls : Chlorophuit ‘a’ is found in all the oxygen, evolving photosynthetic plants except photosynthetic bacteria., Reaction centre of photosynthesis is formed of chlorophyll a. It, occurs in several spectrally distinct forms which perform distinct, roles in photosynthesis (e.g, Chl djgy OF Pray Chl Gzyp OF Pra, etc)., It directly takes part in photochemical reaction. Hence, it is termed, as primary photosynthetic pigment. Other photosynthetic pigments, including chlorophyll b, c d and e ; carotenoids and phycobilins, are called accessory pigments because they do not directly take, part in photochemical act. They absorb specific wavelenaths of, light and transfer energy finally to chlorophyll a through electron, spin resonance., , Chlorophyll @ is bluish-green while chlorophyll b is olivegreen. Both are soluble in organic solvents like alcohol, acetone, etc. Chlorophyll is a green pigment because it does not absorb, green light (but reflect green light) Chlorophyll a (C.H;:0,N,Ma), possesses — CH, (methyl group), which is replaced by — CHO, (an aldehyde) group in chlorophyll b (CzsHjO.NaMg). Chlorophyll, molecule is made up of a squatish tetrapyrrolic ring known as head, and a phytol alcohol called tail. The magnesium atom is present in, the central position of tetrapyrrolic ring. The four pyrrole rings of, Porphyrin head are linked together by methine (CH =) groups, forming a ring system., , When central Mg is replaced by Fe, the chlorophyll becomes a, green pigment called ‘cytochrome’ which is used in photosynthesis, (Photophosphorylation) and respiration both., , , , (2) Carotenoids : They are sometimes called lipochromes, due to their fat soluble nature. They are lipids and found in nongreen parts of plants. Light is not necessary for their biosynthesis., Carotenoids mainly absorb violet, indigo and blue wavelength of, spectrum in higher plants and transfer it to Chl. @ and thus act as, accessory pigments. They protect the chlorophyll molecules from, Photo-oxidation by picking up nascent oxygen and converting it, into harmless molecular stage. Carotenoids can be classified into, two groups namely carotenes and xanthophyll, , (i) Carotenes : They are orange red in colour and have, general formula C,oH.;. They are isolated from carrot., , ‘They are found in all groups of plants i.e., from algae to, angiosperms. Some of the common carotenes are & f, yand &, carotene; phytotene, lycopene, neurosporene etc. The lycopene is, a red pigment found in ripe tomato and red pepper fruits. The 2carotene on hydrolysis gives vitamin A, hence the carotenes are, also called provitamin A. ficarotene is black yellow pigment of, carrot roots, , (ii) Xanthophylls : They are yellow coloured carotenoid also, called xanthols or carotenols. They contain oxygen also along with, carbon and hydrogen and have general formula CHO,, , Lutein (CigHs,0,) a widely distributed xanthophyll which is, responsible for yellow colour in autumn foliage. Fucoxanthin, (CighO.) is another important xanthophyll present in, Phaeophyceae (Brown algae),, , (3) Phycobilins : These pigments are mainly found in bluegreen algae (Cyanobacteria) and red algae. These pigments have, ‘open tetrapyrrolic in structure and do not bear magnesium and, phytol chain,, , , , Blue-green algae have more quantity of phycocyanin and red, algae have more phycoerythrin, Phycocyanin and phycoerythrin, together form phycobilins. These water soluble pigments are, thought to be associated with small granules attached with, lamellae. Like carotenoids, phycobilins are accessory pigments i.e,,, they absorb light and transfer it to chlorophyll a., , Nature of light : Sunlight is a type of energy called radiant, energy or electromagnetic energy. This energy, according to, electromagnetic wave theory (Proposed by James Clark Maxwell,, 1960), travels in space as waves. The distance between the crest of, two adjacent waves is called a wavelength (2). Shorter the, wavelength greater the energy., , The unit quantity of light energy in the quantum theory is, called quantum (hv), whereas the same of the electromagnetic field, is called photon. Solar radiation can be divided on the basis of, wavelengths. Radiation of shortest wavelength belongs to cosmic, rays whereas that of longest wavelength belong to radio waves., Visible light lies between wavelengths of ultra-violet and infra-red,, ‘The visible spectrum of solar radiations are primarily absorbed by, carotenoids of the higher plants are violet and blue. However, out, of blue and red wavelengths, blue light carry more energy., , , , Shortest wavelength ——+ Longest wavelength, Maximum ene Minimum enery, Visible light : 390nm (3900A) to 760nm (7600A). Violet, (390-430nm), blue (430-470nm), blue-green (470-500nm), aren, (500-580nm), yellow (580-600nm), orange (600-650nm),, orange-red (650-660nm) and red (660-760nm) Far-red (700760nm). Infra-red 760nm ~ 100um. Ultraviolet 100-390nm. Solar, radiations 300nm (ultraviolet) to 2600nm (infra-red).

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658 Photosynthesis in Higher Plants:, , , , , , , , Photosynthetically active radiation (PAR) is 400-700nm. Leaves, appear green because chlorophylls do not absorb green light. The, same is reflected and transmitted through leaves., , Absorption and action spectra : The curve representing, the light absorbed at each wavelength by pigment is called, absorption spectrum. Curve showing rate of photosynthesis at, different wavelengths of light is called action spectrum., , 180, 160, , , , 140, 120, , , , 100, 80, 60, 40, 20, , Specific absorption, , , , TT, , , , , , sti 2 LS ENE, 380 420 460 500 540 580 620 660, Wavelength, m jt, Fig : 4.3-1 Absorption spectra of chlorophylls a and b, Absorption spectrum is studied with the help of, spectrophotometer. The absorption spectrum of chlorophyll a and, chlorophyll b indicate that these pigments mainly absorb blue and, red lights. (430 nm and 662 nm for chlorophyll a, 455 nm and 644, nm for chlorophyll b). Action spectrum shows that maximum, photosynthesis takes place in blue and red regions of spectrum., The first action spectrum of photosynthesis was studied by T.W., Engelmann (1882) using green alga Spirogyra and oxygen seeking, bacteria,, , In this case actual rate of photosynthesis in terms of oxygen, evolution or carbon dioxide utilisation is measured as a function of, wavelength., , Mechanism of photosynthesis, , On the basis of discovery of Nicolas de Saussure that "The, amount of O; released from plants is equal to the amount of CO,, absorbed by plants’, it was considered that O, released in, photosynthesis comes from CO;, but Ruben proved that this, concept is wrong,, , In 1930, C.B. Van Niel proved that, sulphur bacteria use H_S, {in place of water) and CO, to synthesize carbohydrates as follows:, , , , , , 6CO, +12H,S—+C,H,0, +6H,0+125, , This led Van Niel to the postulation that in green plants, water, (H,0) is utilized in place of H,S and O, is evolved in place of, sulphur (S). He indicated that water is an electron donor in, photosynthesis., , 6CO; + 12H30—> CH 205 + 620 + 602, , This was confirmed by Ruben and Kamen in 1941 using, Chlorella a green alga., , , , They used isotopes of oxygen in water, ie, H,"0 instead of, H,0 (normal) and noticed that liberated oxygen contains "O of, water and not of CO;, The overall reaction can be given as under =, , 6CO, +12H,"0 CgHy,Og +60, +6HzO, , During photosynthesis the OQ, in glucose comes from, carbondiowater., , Modern concept of photosynthesis, , Photosynthesis is an oxidation reduction process in which, water is oxidised to release O, and CO, is reduced to form starch, and sugars., , Scientists have shown that photosynthesis is completed in, two phases., , (1) Light phase or Photochemical reactions or Light, dependent reactions or Hill's reactions : During this stage, energy from sunlight is absorbed and converted to chemical energy, which is stored in ATP and NADPH + H*., , (2) Dark phase or Chemical dark reactions or Light, Independent reactions or Blackman reaction or, Biosynthetic phase : During this stage carbohydrates are, synthesized from carbon dioxide using the energy stored in the, ATP and NADPH formed in the light dependent reactions., , Evidence for light and dark reactions in photosynthesis, , (1) Physical separation of chloroplast into grana and, stroma fractions : It is now possible to separate grana and, stroma fractions of chloroplast. If light is given to grana fraction in, presence of suitable H-acceptor and in complete absence of CO;,, then ATP and NADPH, are produced (Le,, assimilatory powers). If, these assimilatory powers (ATP and NADPH,) are given to stroma, fraction in presence of CO, and absence of light, then, carbohydrates are formed., , (2) Experiments with intermittent light or Discontinuous, light : Rate of photosynthesis is faster in intermittent light, (Alternate light and dark periods) than in continuous light. It is, because light reaction is much faster than dark reaction, so in, continuous light, there is accumulation of ATP and NADPH, and, hence reduction in rate of photosynthesis but in discontinuous, light, ATP and NADPH, formed in light are fully consumed during, dark in reduction of CO; to carbohydrates. Accumulation of, NADPH, and ATP is prevented because they are not produced, during dark periods, , (3) Temperature coefficient studies : Blackman found, that Qj, was greater than 2 in experiment when photosynthesis, ‘was rapid and that Qyo dropped from 2 often reaching unity, Le. 1, when the rate of photosynthesis was low. These results show that, in photosynthesis there is a dark reaction (Q) more than 2) and a, photochemical or light reaction (with Q,, being unity)., , Reaction rate of (t +10)°C, Reaction at t*C, , , , Qo =

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Photosynthesis in Higher Plants 659, , , , , , Light reaction (Photochemical reactions) : Light, Feaction occurs in grana fraction of chloroplast and in this reaction, are included those activities, which are dependent on light., Assimilatory powers (ATP and NADPH,) are mainly produced in, this light reaction,, , Robin Hill (1939) first of all showed that if chloroplasts, extracted from leaves of Stellaria media and Lamium album are, ‘suspended in a test tube containing suitable electron acceptors,, g,, Potassium ferroxalate (Some plants require only this, chemical) and potassium ferricyanide, oxygen is released due to, photochemical spliting of water. Under these conditions, no CO,, was consumed and no carbohydrate was produced, but lightdriven reduction of the electron acceptors was accompained, by O,, evolution., , 4Fe™*+2H,0:—4Fe**+ 4H" +0, 7, corer Sete Prat, , scoper Heston, , The splitting of water during photosynthesis is called, photolysis. This reaction on the name of its discoverer is known as, Hill reaction,, , Hill reaction proves that, (1) In photosynthesis oxygen is released from water., , (2) Electrons for the reduction of CO, are obtained from water, Iie, a reduced substance (hydrogen donor) is produced which, later reduces CO,)., , Dichlorophenol indophenol is the dye used by Hill for his, famous Hill reaction,, , ‘According to Amon (1961), in this process light energy is, converted to chemical energy. This energy is stored in ATP (this, Process of ATP formation in chloroplasts is known as, photophosphorylation) and from electron acceptor NADP*, a, substance found in all living beings NADP*H is formed as, hydrogen donor. Formation of hydrogen donor NADPH from, electron acceptor NADP* is known as photoreduction or, Production of reducing power NADPH., , Light phase can be explained under the following, headings, , (1) Transfer of energy : When photon of light energy falls, on chlorophyll molecule, one of the electrons pair from ground, or singlet state passes into higher energy level called excited, singlet state. It comes back to hole of chlorophyll molecule, within 10? seconds., , This light energy absorbed by chlorophyll molecule before, coming back to ground state appears as radiation energy, while, that coming back from excited singlet state is called fluorescence, and is temperature independent. Sometimes the electron at excited, singlet state gets its spin reversed because two electrons at the, ‘same energy level cannot stay; for sometime it fails to return to its, Partner electron. As a result it gets trapped at a high energy level, , Due to little loss of energy, it stays at comparatively lower energy, level (Triplet state) from excited singlet state. Now at this moment,, it can change its spin and from this triplet state, it comes back to, ground state again losing excess of energy in the form of radiation,, This type of loss of energy is called as phosphorescence., , Photon nal, flight Orig, [ne ook, , (@) —, , Ground state, , , , , (a) Excited state, , Excited second singlet state, Heat, Excited first singlet state, , , , , Internal, , (Phosphorescence), , 6), Ground state °), , Fig : 4.3-2 (a) Photoexcitation of chlorophyll, molecule i.c., of its atoms (b) Movement of electron, due to photoexcitation of pigment molecule, When electron is raised to higher energy level, it is called at, second singlet state, It can lose its energy in the form of heat also., Migration of electron from excited singlet state to ground state, along with the release of excess energy into radiation energy is of, no importance to this process. Somehow when this excess energy, is converted to chemical energy, it plays a definite constructive role, in the process., (2) Quantum yield, (i) Rate or yield of photosynthesis is measured in terms of, quantum yield or O, evolution, which may be defined as, "Number, of O, molecules evolved per quantum of light absorbed in, photosynthesis.", (ii) Quantum requirement in photosynthesis =, quanta of light are required to evolve one mol. of O;., , 8, Le, B, , (iil) Hence quantum yield = 1/8 = 0.125 (Le,, a fraction, of 1) as 12%., , (3) Emerson effect and Red drop : R, Emerson and, CM. Lewis (1943) observed that the quantum yield of, Photosynthesis decreases towards the far red end of the

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660 Photosynthesis in Higher Plants, , , , , , spectrum (680nm or longer). Quantum yield is the number of, oxygen molecules evolved per light quantum absorbed. Since, this decrease in quantum yield is observed at the far region or, beyond red region of spectrum is called red drop., , Emerson et al, (1957) further observed that photosynthetic, efficiency of light of 680nm or longer is increased if light of shorter, wavelengths (Less than 680nm) is supplied simultaneously. When, both short and long wavelengths were given together the quantumyield of photosynthesis was greater than the total effect when both, the wavelengths were given separately. This increase in, photosynthetic efficiency (or quantum yield) is known as Emerson, effect or Emerson enhancement effect., , , , , , Quantum yieldin combined beam—Quantum yieldin red beam., Quantum yieldin farred beam, , (4) Two pigment systems : The discovery of Emerson effect, has clearly shown the existence of two distinct photochemical, processes, which are believed to be associated with two different, specific group of pigments., , (i) Pigment system I or Photosystem I : The important, pigments of this system are chlorophyll a 670, chlorophyll a 683,, chlorophyll a 695, Pro. Some physiologists also include carotenes, and chlorophyll b in pigment system I. Pr acts as the reaction, centre. Thus, this system absorbs both wavelengths shorter and, longer than 680nm., , , , (i) Pigment system Il or photosystem Il : The main, pigments of this system are chlorophyll a 673, Pigp, chlorophyll b, and phycobilins. This pigment system absorbs wavelengths shorter, than 680nm only, Pin acts as the reaction centre., , Pigment system I and Il are involved in non-cyclic electron, transport, while pigment system 1 is involved only in cyclic, electron transport. Photosystem I generates strong reductant, NADPH. Photosystem Il produces a strong oxidant that forms, oxygen from water., , Table : 4.3-1 Comparison of photosystem I and photosystem II, , , , , , , , , , ‘S.No, | _ Photosystem 1 Photosystem II, @) [PS 1 lies on the outer | PS Il lies on the inner surface, surface ofthe thylakoids. | of the thylakoid., (2) _| im this system molecular ||As the result of photolais of, oxygen isnot evolved. | water molecular oxygen is, evolved., (3) [lis reaction center is | Ils reaction center is PO8O,, , P70., (4) |/W pamicipates both in, , , , If participates only in noneyclic, , gyclic and noneyclic | photophasphorylation,, photophosphorylation, (5) | it Teceives electrons | It receives electrons from, , from photosystem il. | photolytic dissociation of, , , , , , water., © (6) |i is not related with | Its related with photos of, photolysis of water. water., , , , (5) Photophosphorylation : Light phase includes the, interaction of two pigment systems. PS I and PS Il constitute, various type of pigments. Amon showed that during light reaction, not only reduced NADP is formed and oxygen is evolved but ATP, is also formed. This formation of high energy phosphates (ATP) is, dependent on light hence called photophosphorylation., , Photophosphorylation is of two types, , (i) Cyclic photophosphorylation : The system is found, dominantly in bacteria, It involves only PS 1. Flow of electron is, cyclic, NADP is not available then this process will occur. When, the photons activate PS I, a pair of electrons are raised to a, higher energy level. They are captured by primary acceptor, which passes them on to ferredoxin, plastoquinone, cytochrome, complex, plastocyanin and finally back to reaction centre of PS |, lLe., Pro. At each step of electron transfer, the electrons lose, potential energy. Their trip down hill is caused by the transport, chain to pump H” across the thylakoid membrane. The proton, gradient, thus established is responsible for forming (2 molecules), ATP. No reduction of NADP to NADPH+ H". ATP is synthesized, at two steps., , , , Reducing, 8, &, 5, , , , ADP+IP, , , , , , , , , , Redox potential, 4, is, , , , Oxidizing, , Antenna, ‘molecules, , Fig : 4.3-3 Cyclic photophosphorylation, , , , Non cyclic photophosphorylation : The system is, dominant in green plants. It involves both PS-I and PS-II. Flow of, electrons is unidirectional. Here electrons are not cycled back and, are used in the reduction of NADP to NADPH,. Here H,0 is, utilized and O, evolution occurs. In this chain high energy electrons, released from 'P-680' do not return to 'P-680' but pass through, pheophytin, plastoquinone, cytochrome bef complex, plastocyanin, (Cu containing pigment) and then enter P-700, In this transfer of, electrons from plastoquinone (PQ) to cytochrome bef complex,, ATP is synthesized, Because in this process high energy electrons, released from 'P-680' do not retum to 'P-680' and ATP (1, molecules) is formed, this is called Nncyclic, photophosphorylation. ATP is synthesized at only one step.