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Photosynthesis chlorophyll is the pigment that permits: photosynthesis 6CO2 + 6H2O + light energy C6H12O6 + 6O2 6CO2 + 6H2O C6H12O6 + 6O2 this reaction is not spontaneous! CO2 + H2O H2CO3 •Photosynthesis is possible thanks to chlorophyllcontaining vegetal organisms that are capable of absorbing sunlight and of converting it into energy contained in the chemical bounds of organic molecules. •Photosynthesis is the most important process occurring in Earth in terms of production of organic matter and of maintenance of life. •To have an idea of how productive is this process (in terms of organic matter), we could imagine a stack of books covering 17 times the distance between Earth and Sun. This corresponds to 160 billion tons of carbohydrates, and is the annual production of the process. •At the beginning of its evolution, terrestrial atmosphere was similar to that of Mars and Venus (predominance of CO2 against O2). Photosynthesis permitted a change in the composition, by increasing the oxygen content with consequent formation of ozone (O3) which allowed a protection against the destructive ultraviolet rays. The increasing level of O2 permitted the evolution of aerobic organisms using energy of organic compounds produced by photosynthesis. What would happen if photosynthesis reduced? •About 65 millions years ago, a meteorite crashed upon Earth causing a huge cloud of dust that absorbed sunlight and reduces photosynthesis and so plant growth and survival. •As a consequence, there was the extinction of dinosaurs that permitted evolution and diffusion of mammals. Photosynthesis and secondary metabolites •As a consequence of the increase of atmospheric oxygen, plant produced more oxygenated compounds acting as scavengers of the harmful free radicals produced by UV rays. Photosynthesis vs Respiration Photosynthesis vs Respiration 6O2 •Organic compounds are synthesized by the reduction of carbon dioxide using energy absorbed by chlorophyll from sunlight. •3 CO2 + 6 H2O light C3H6O3 + 3O2 + 3 H2O •In green plants, water acts as both a hydrogen donor and a source of released oxygen. •The leaf mesophyll is the site of photosynthesis. O2, CO2 and water vapor enter and exit through stomata. photosynthetic organisms •Photosynthetic bacteria are unable to utilize water and therefore do not produce oxygen. Instead the may use hydrogen sulphide (purple and green sulphur bacteria) as a source of hydrogen. As a consequence, sulphur (giving yellow colour to bacteria) is accumulated in cells. CO2 + 2H2S luce (CH2O) + H2O + 2S Nature of the light and role of the photosynthetic pigments •The visible light is a part of a wide spectrum of radiations emitted by sun, the electromagnetic spectrum, that is composed of radiations emitted into free space under different wavelegnths (λ) which are perceived as the different colours (Maxwell wave model, 1831-1879). The higher is the wave lenght (λ) of a radiation, the lower is its frequency (ν) and vice versa (inversely proportional). •Terrestrial atmosphere acts as a selective filter allowing passing of part of the electromagnetic spectrum, known as visible light that is used by photosynthesis. The visible spectrum comprises radiations with λ ranging from 380 nm (ultraviolet light) to 750 nm (red light). •When the light hits an object, some wavelenghts are absorbed while others are reflected or transmitted. The perception of the various colours is determined by the wavelenght of the reflected or transmitted radiation by the object. The human eye is sensitive only to radiations with λ in the range of 380-750 nm. Corpuscolar theory (Einstein, 1905) •The light may be thought also as a set of energy particles known as photons. •The energy of a photon is given by E=hν, where ν is the frequency of the radiation that is directly proportional to the energy and inversely proportional to the wavelenght (λ) of the photon; h is the Plank’s constant. •Therefore, the violet photons have about double energy respect to the red photons, and about half their wavelenght. Photosynthetic pigments •They are light-abosrbing molecules taking part to photosynthesis. •They occur on thylakoids of chloroplasts of plant cell, or on special membranes of photosynthetic bacteria and blue-green algae. •A pigment absorbs only a particular wavelenght and transmits or reflects light of a different wavelenght. The type of wavelenghts absorbed or reflected by a pigment represents its absorbance spectrum. •Chlorophylls absorb light of violet, blue and red wavelenght and reflect green wavelenght so that they appear as green. Absorbance spectrum Absorbance spectrum chlorophyll red blood cells •When a pigment absorbs a photon, its electrons are pushed to a higher energy level in about 10-15 sec. (the excited state, highly instable), from which then they get back to the lower energy level in about 10–8 sec. (the ground state, stable) by releasing the absorbed energy in 3 possible ways: 1) Energy is converted into a combination of heat and light with a higher wavelenght (fluorescence); this phenomen happens only when chlorophyll has been isolated from tylakoids (e.g. in a solution). 2 3 2) Energy but not electrons are transferred from a chlorophyll to another so that the latter be excited and the first one backs to the ground state. This kind of conversion is called resonance energy transfer. 3) Excited electron of the pigment is transferred to an adjacent molecule known as primary electron acceptor, that is normally a component of the so-called electron transport chain, so that the pigment lacks an electron (i.e. is oxidized). •The only photons to be absorbed by pigments are those having an energy equal to the difference between the excited state and the ground state. This difference varies from an atom or molecule to another. •Such a compound may absorb only photons of a specific wavelenght, that’s why they have a peculiar absorbance spectrum. Photosynthetic pigments •Chlorophyll a is the fundamental pigment for photsynthesis in eukaryotic organisms and cyanobacteria; it constitutes about 75% of the total chlorophyll in higher plants, responsible for the green colour. •Accessory pigments (belonging to the complex antenna) are not directly involved in the light phase; they increase the energy spectrum utilizable by chlorophyll a by absorbing such wavelenghts (e.g. 440-470 nm) and then transfering them to the chlorophyll a which in turn convert into chemical energy: •chlorophyll b (occurring in plants and green algae). •chlorophyll c (occurring in brown algae). •chlorophyll d (occurring in red algae). •carotenoids such as carotenes and xanthophylles, soluble in lipids (giving red, orange and yellow colours); they protect the cell protoplast from photooxidation and photodestruction (they act as antioxidant). •phycobilins (occurring in red algae and cyanobacteria), soluble in water. Correlation between action spectrum of photosynthesis and absorbance spectrum of chlorophyll Correlation between action spectrum of photosynthesis and absorbance spectrum of chlorophyll •The action spectrum represents the efficacy of different wavelenghts on photosynthesis (in terms of rate or oxygen production). The correlation between the action spectrum of a light-dependent process and the absorbance spectrum of a pigment, shows that that pigment is mostly involved in that process. •The similarity between the absorbance spectrum of chlorophyll a and the action spectrum of photosynthesis is the evidence that chlorophyll a is the main pigment involved in photosynthesis. Correlation between action spectrum of photosynthesis and absorbance spectrum of chlorophyll 6 CO2 + 6 H2O luce C6H12O6 + 6O2 3 CO2 + 6 H2O luce C3H6O3 + 3O2 + 3 H2O Photosynthesis phases The energy yield is about 92%! Photosynthesis happens in 2 phases: 1. Light reactions. 2. Calvin cycle (or dark phase). 1) They occur on tylakoids; the light energy absorbed by chlorophyll is used to synthesize ATP (adenosintriphosphate). At the same time water is dissociated in oxygen, electrons and protons leading to the production of NADPH (nicotinamide adenine dinucleotide phosphate). 2) They occur in the stroma where ATP and NADPH, produced during light reactions, are used to synthesize organic molecules (e.g. glucose, sucrose, starch, secondary metabolites etc.) from glyceraldehyde 3-phosphate(PGAL). nicotinamide ribose adenine ribose ATP NADP •The two molecules in which the chemical energy, converted from the light by chlorophyll, is stored. •The process that produces them is known as photophosphorilation. •Starch grains (primary starch) in a chloroplast 1) Photosystem •A light-harvesting unit of photosynthesis that, in green plants, comprises about 300 light-absorbing molecules (the so called complex antenna made of accessory pigments) linked to the tilakoid membranes, with a molecule of chlorophyll a acting as the reaction centre. It is involved in the light reactions of photosynthesis. Internal surface of a tylakoid at SEM Photosystem 1) •Light absorbed by a pigment, by resonance energy transfer, passes from a molecule to another in the complex antenna up to be collected by one of the two chlorophyll a molecules belonging to the rection center where it is converted in chemical energy (ATP and NADPH). Photosystems 1) There are 2 different types of photosynthetic units: Photosystem I (PSI) (discovered first): involving the pigment P700 (a special chlorophyll a molecule), chlorophyll b, and accessory pigments, that requires light of longer wavelenght than photosystem II; it results in the reduction of NADP+ and the production of ATP through photophosphorylation. Photosystem II (PSII) (discovered aftewards): involving the pigment P680 (a special chlorophyll a molecule), chlorophyll b, and accessory pigments, that requires light of shorter wavelenght than photosystem I; it results in the dissociation of water and the evolution of molecular oxygen. Non-cyclic photophosphorylation 1) •The electron transfer in the electron transport chain between the 2 PS is strictly connected with the proton transfer across the thylakoid membrane, determining a proton gradient which is fundamental for the synthesis of ATP. In addition the reduction of NADP happens at the end of the process. electron transport chain I electron transport chain II photolysis •Electron follow the way: water -> PSII -> electron transport chain I > PSI -> electron transport chain II -> NADP+. Non-cyclic photophosphorylation 1) ATP-synthase are the only points of the membrane freely permeable to protons (H+) Non-cyclic photophosphorylation 1) H •The elctron transport chain is arranged such that it generates an energy-rich proton gradient across the thylakoid membrane. This energy is then used to drive the phosphorilation of ADP through a membrane-bound ATP-synthase. Chemiosmotic photophosphorilation 1) Cyclic photophosphorylation 1) •Cyclic photophosphorilation is the synthesis of ATP during photosynthesis, coupled to the cyclic passage of electrons to and from P700, the specialized form of chlorophyll a which is involved in PSI, using a series of carrier molecules. Neither O2 nor NADPH are produced during this process. The process is needed to support the energy demand of the Calvin cycle that consumes more ATP than NADPH. 2) •Across stomata, the CO2 needed for Calvin cycle (dark phase) spreads inside the mesophyll. At the same time, oxygen produced during light phase spreads outside. 2) 3-phosphoglycerate kinase STROMA •3 turns are needed to produce one molecule of glyceraldehyde 3phosphate (C3H6O3) o ciclo C3 glyceraldehyde 3-phosphate deidrogenase Calvin cycle (C3 pathway) RuBisCO (RuBP carbossilasi/ossigenasi) Fixation of CO2 by RuBisCO 2) •RuBisCO (ribulose biphosphate carboxylase/oxygenase) is the most abundant enzyme in the world. 2) Calvin cycle (C3 pathway) •It is a cyclic series of reactions occurring in the stroma of chloroplasts, in which carbon dioxide is fixed and reduced to glucose, using ATP and NADPH formed in the light reaction of photosynthesis. The relatively stable product formed is a 3carbon sugar. •Phase I: CO2 is fixed to ribulose biphosphate (RuBP) by RuBisCO forming 3phosphoglycerate (fixation). •Phase II: reduction of 3-phosphoglycerate to glyceraldehyde 3-phosphate (G3P) using ATP and NADPH (reduction). •Phase III: 5 of the 6 molecules of glyceraldehyde 3-phosphate (synthesized after 3 cycles) are used to regenerate ribulose biphosphate (RuBP), the starting compound, using ATP, while the reamining molecule is used to synthesize new organic compound (e.g. glucose, sucrose, starch, secondary metabolites etc.) (regeneration). •Each reaction is catalyzed by a specific enzyme. To synthesize a molecule of glyceraldehyde 3-phosphate are needed 3 molecules of carbon dioxide (3 turns of the cycle); more ATP (9 mol.) than NADPH (6 mol.) is consumed (that is the reason of the need of a cyclic photophosphorilation). 2) •Indeed photosynthetic cells produce low levels of glucose. •Most of G3P synthesized by Calvin cycle is exported in cytoplasm where it may be converted into sucrose (the main form of sugar transport in plants). •G3P remaining in the chloroplast is temporarely converted into primary starch to avoid osmotic damage. At night, starch is hydrolysed to produce sucrose that is then exported from the leaf to the other parts trough the phloem. Pharmaceutical importance of sucrose •Sucrose (saccharose) is used as sweetener, nutritional supplement, and as carrier and preservative agent (at high concentrations it inhibites the growth of organisms). Oxigenase activity of RuBisCO •RuBisCO is unable to discriminate between CO2 and O2, so that in some conditions (e.g. arid climates) it may have oxygenase activity. In this case it produces 1 molecule of 3-phosphoglycerate (directed to the Calvin cycle) and 1 molecule of 2phosphoglycolate that is a non-useful metabolite (i.e. does not give rise to organic compounds). As a consequence of the RuBisCO oxygenase activity, plant cell recycles this metabolite through a series of reactions known as photorespiration, that consume ATP and eliminate carbon atoms (i.e. reduction of photosynthetic yield). Photorespiration •Photorespiration is a light-activated type of respiration that occurs in plant cell. It differs from normal respiration in that it involves glycolate metabolism. It is a process that is wasteful of both carbon dioxide and energy. •Photorespiration is given by the oxygenase activity of RuBisCO together with the pathway of phosphoglycolate recycle. During this process oxygen is consumed while carbon dioxide is eliminated leading to a decrease of photosynthetic yield (i.e. plant synthesize less organic matter). This process involves chloroplast, peroxisome and mithocondrion. Photorespiration •During photorespiration, oxygen is consumed while carbon dioxide is eliminated so that the plant has less carbon atoms to use in the synthesis of organic compounds (Calvin cycle). With respect to cell respiraton, the process do not produce ATP and NADH. Conditions favouring photorespiration: High temperatures and hydric stress leading to closing of stomata. Low gradient of carbon dioxide due due to the high plant density (e.g. in rain forests). How we can explain photorespiration? •It has been hypothesize that photorespiraton is a relic of evolution, since RuBisCO has remained equal to that occurring when the atmospheric composition was quite different. At that time (e.g. 4 billions years ago) the incapacity of RuBisCO to discriminate between CO2 and O2 did not produce significant level of photorespiration as much as at present, owing to the scarcity of oxygen in the atmosphere. Alternative processes of carbon fixation •Tropical plants adapted to high temperatures, strong light, low carbon dioxide levels, and low water supply evoluted an alternative pathway of carbon fixation known as C4 pathway. Its importance lies mainly in its reduction of photorespiration. •The first product formed as a result of the carboxylation by CO2 of an acceptor molecule, phospho-enolpyruvate (PEP) is the four-carbon oxaloacetate (OAA). This reaction is catalyzed by the enzyme PEP carboxylase occurring in the cytoplasm. Eventually the CO2 is handed on to, and refixed by, the Calvin cycle. •The 2 most important alternative pathways are the C4 metabolism and the crassulacean acid metabolism (CAM). C4 pathway C4 pathway reduces photorespiration! •PEP carboxylase is a more efficient enzyme to fix CO2 (as bicarbonate ion) so that it is capable to function even if stomata are almost closed due to environmental conditions. C4 pathway •Initial carboxylation occurs in mesophyll (M) cells via PEP carboxylase (PEPC) catalysing the formation of oxaloacetate (OAA) from bicarbonate and phospho enolpyruvate (PEP). OAA is converted into malate in M chloroplasts then malate diffuses into bundle sheath (BS) cells via plasmodesmata. Decarboxylation occurs in BS chloroplasts increasing the CO2 concentration in the BS to 1–2%. The increased CO2 concentration suppresses the oxygenation activity of RuBisCO. The decarboxylation product, pyruvate, is regenerated into the primary carbon acceptor PEP in the M chloroplasts. C4 pathway Spatial separation between C4 (into mesophyll cells) and Calvin Cycle (into bundle-sheat bundle cells) pathways. C4 pathway •Plawsmodesmata through which 4-carbon metabolites pass from mesophyll cell to bundle sheath cell. C4 pathway % distribution of C4 plant in the north America. They increase in number in warm regions. The advantages of the C4 pathway • C4 plants have a competitive advantage over plants possessing the more common C3 carbon fixation pathway under conditions of drought, high temperatures, and nitrogen or CO2 limitation. • When grown in the same environment, at 30°C, C3 grasses lose approximately 833 molecules of water per CO2 molecule that is fixed, whereas C4 grasses lose only 277 water molecules per CO2 molecule fixed. This increased water use efficiency of C4 grasses means that soil moisture is conserved, allowing them to grow for longer in arid environments. • About 7600 species of plants use C4 carbon fixation, which represents about 3% of all terrestrial species of plants. All these 7600 species are angiosperms (mainly monocotyledones). CAM pathway •CAM pathway: carbond dioxide is fixed during night when stomata are open. Starch is hydrolyzed to phosphoenolpyruvate that fixes CO2 in the cytosol forming malate that enter vacuole as malic acid. During day malate come out from vacuole and is decarboxylated; CO2 enter chloroplast where it is used in the Calvin cycle, while pyruvate give back starch by an inverse glycolysis. CAM pathway •In a plant using full CAM, the stomata in the leaves remain closed during the day to reduce evapotranspiration, but open at night to collect carbon dioxide (CO2). •During the night, stomata are open, allowing CO2 to enter and be fixed as organic acids that are stored in vacuoles. During the day the stomata are closed (thus preventing water loss), and the carbon is released to the Calvin cycle so that photosynthesis may take place. •The carbon dioxide is fixed in the cytoplasm of mesophyll cells by a PEP reaction similar to that of C4 pathway. But, unlike the C4 mechanism, the resulting organic acids are stored in vacuoles for later use; that is, they are not immediately passed on to the Calvin cycle. The latter cannot operate during the night because the light reactions that provide it with ATP and NADPH cannot take place during the night, the CO2-storing organic acids are released from the vacuoles of the mesophyll cells and enter the stroma of the chloroplasts where an enzyme releases the CO2, which then enters into the Calvin cycle. Differences between C4 and CAM metabolisms •In C4 plant (e.g. corn, sugar cane etc.) fixation of carbon dioxide and calvin cycle happen in different cells (spatial separation between C4 and C3 pathways). Stomata are open by day and closed by night. •In CAM plant (e.g. Cactaceae and Crassulaceae) fixation of carbon dioxide and calvin cycle happen in the same cell, the former by night, the latter by day (temporal separation between C4 and C3 pathways). Stomata are open by night and closed by day.