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 CO₂ fixed annually through photosynthesis is about 7.0 × 10¹³kg. Photosynthesis is an anabolic and endothermic reaction. Photosynthesis helps to maintain the equilibrium position of O₂ and CO₂ in the atmosphere.  

1. HISTORICAL BACKGROUND
Before seventeenth century it was considered that plants take their food from the soil.

• Van Helmont (1648) concluded that all food of the plant is derived from water and not from soil.
• 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 (e., Phlogiston), produced by burning of candle, and convert it into pure air (i.e., Dephlogiston).
• Jan Ingen-Housz (1779) concluded by his experiment that purification of air was done by green parts of plant only and that too in the presence of sunlight. Green leaves and stalks liberate dephlogisticated air (Having O₂) during sunlight and phlogisticated air (Having CO₂) during dark.
• Jean Senebier (1782) proved that plants absorb CO₂ and release O₂ in presence of light. He also showed that the rate of O₂ evolution depends upon the rate of CO₂
• Nicolus de Saussure (1804) showed the importance of water in the process of photosynthesis. He further showed that the amount of CO₂ absorbed is equal to the amount of O₂
• 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 CO₂.
• Bousingault (1860) reported that the volume of CO₂ absorbed is equal to volume of O₂ evolved and that CO₂ absorption and O₂ evolution get start immediately after the plant was exposed to sunlight.
• Julius Von Sachs (1862) demonstrated that first visible product of photosynthesis is starch. He also showed that chlorophyll is confined to the chloroplasts.
• Melvin Calvin (1954) traced the path of carbon in photosynthesis (Associated with dark reactions) and gave the C₃ 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.

2. PHOTOSYNTHESIS IN HIGHER PLANTS
Chloroplast (The site of photosynthesis)
Chloroplast are green plastids which function as the site of photosynthesis in eukaryotic photoautotrops.
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 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. 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:
(1) Chlorophylls: Chlorophyll ‘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 a₆₈₀ or P₆₈₀, Chl a₇₀₀ or P₇₀₀, 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 wavelengths of light and transfer energy finally to chlorophyll a through electron spin resonance.
Chlorophyll a is bluish-green while chlorophyll b is olive-green. 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 (C₅₅H₇₂O₅N₄Mg) possesses — CH₃ (methyl group), which is replaced by — CHO (an aldehyde) group in chlorophyll b (C₅₅H₇₀O₆N₄Mg). Chlorophyll molecule is made up of a squarish 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 non-green parts of plants. Light is not necessary for their biosynthesis. Carotenoids absorb light energy and transfer it to Chl. a 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 C₄₀H₅₆. 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 α, β, γ and δ carotene; phytotene, lycopene, neurosporene etc. The lycopene is a red pigment found in ripe tomato and red pepper fruits. The β-carotene on hydrolysis gives vitamin A, hence the carotenes are also called provitamin A. β-carotene is black yellow pigment of carrot roots.
(ii) Xanthophylls: They are yellow coloured carotenoid also called xanthols or carotenols. They contains oxygen also along with carbon and hydrogen and have general formula C₄₀H₅₆O₂.
Lutein (C₄₀H₅₆O₂) a widely distributed xanthophyll which is responsible for yellow colour in autumn foliage. Fucoxanthin (C₄₀H₅₆O₆) is another important xanthophyll present in Phaeophyceae (Brown algae).
(3) Phycobilins: These pigments are mainly found in blue-green 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 (λ). Shorter the wavelength greater the energy.
The unit quantity of light energy in the quantum theory is called quantum (hν), 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.
$\underset{Maximum energy}{Shortest wavelength}\to \underset{minimum energy}{Longest wavelength}$

Visible light: 390nm (3900Å) to 760nm (7600Å). Violet (390–430nm), blue (430–470nm), blue-green (470–500nm), green (500–580nm), yellow (580–600nm), orange (600–650nm), orange-red (650–660nm) and red (660–760nm) Far-red (700–760nm). Infra-red 760nm – 100μm. Ultraviolet 100–390nm. Solar Radiations 300nm (ultraviolet) to 2600nm (infra-red). 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.

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. 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.

3. MECHANISM OF PHOTOSYNTHESIS
On the basis of discovery of Nicolas de Saussure that “The amount of O₂ released from plants is equal to the amount of CO₂ absorbed by plants”, it was considered that O₂ released in photosynthesis comes from CO₂, but Ruben proved that this concept is wrong.
In 1930, C.B. Van Niel proved that, sulphur bacteria use H₂S (in place of water) and CO₂ to synthesize carbohydrates as follows:
$6C{O}_2+12H_{2}S\to C_6{H}_{12}{O}_6+6H_{2}O+12S$

This led Van Niel to the postulation that in green plants, water (H₂O) is utilized in place of H₂S and O₂ is evolved in place of sulphur (S). He indicated that water is electron donar in photosynthesis.
$6C{O}_2+12H_{2}O\to C_6{H}_{12}{O}_6+6H_{2}O+6O_2$

This was confirmed by Ruben and Kamen in 1941 using Chlorella a green alga.
They used isotopes of oxygen in water, i.e., H₂¹⁸O instead of H₂O (normal) and noticed that liberated oxygen contains ¹⁸O of water and not of CO₂. The overall reaction can be given as under:
$6C{O}_2+12{H_2}^{18}O\underset{Chlorophyll}{\overset{Light}{\to }}{C}_6{H}_{12}{O}_6+6^{18}{O}_2+6H_{2}O$

4. MODERN CONCEPT OF PHOTOSYNTHESIS
Photosynthesis is an oxidation reduction process in which water is oxidised to release O₂ and CO₂ 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 CO₂, then ATP and NADPH₂ are produced (i.e., assimilatory powers). If these assimilatory powers (ATP and NADPH₂) are given to stroma fraction in presence of CO₂ 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 NADPH₂ and hence reduction in rate of photosynthesis but in discontinuous light, ATP and NADPH₂ formed in light are fully consumed during dark in reduction of CO₂ to carbohydrates. Accumulation of NADPH₂ and ATP is prevented because they are not produced during dark periods.
(3) Temperature coefficient studies: Blackman found that Q₁₀ was greater than 2 in experiment when photosynthesis was rapid and that Q₁₀ dropped from 2 often reaching unity, i.e., 1 when the rate of photosynthesis was low. These results show that in photosynthesis there is a dark reaction (Q₁₀ more than 2) and a photochemical or light reaction (with Q₁₀ being unity).
$Q_{10}=\frac{Reaction rate of (t+10)°C}{Reaction at t°C}$
Light reaction (Photochemical reactions)
Light reaction occurs in grana fraction of chloroplast and in this reaction are included those activities, which are dependent on light. Assimilatory powers (ATP and NADPH₂) 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, e.g., Potassium ferroxalate (Some plants require only this chemical) and potassium ferricyanide, oxygen is released due to photochemical splitting of water. Under these conditions, no CO₂ was consumed and no carbohydrate was produced, but light-driven reduction of the electron acceptors was accompained, by O₂ evolution.

$\underset{Electron acceptor}{4F{e}^{3+}}+\underset{Electron donor}{2H_{2}O}\leftrightarrow \underset{Reduced product}{4F{e}^{2+}}+4H^{+}+O_2 \uparrow$
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 CO₂ are obtained from water [i.e., a reduced substance (hydrogen donor) is produced which later reduces CO₂].
Dichlorophenol indophenol is the dye used by Hill for his famous Hill reaction.
According to Arnon (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^–9 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 some time 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.

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 O₂ evolution, which may be defined as, “Number of O₂ molecules evolved per quantum of light absorbed in photosynthesis.”
(ii) Quantum requirement in photosynthesis = 8, i.e., 8 quanta of light are required to evolve one mol. of O₂.
(iii) Hence quantum yield = 1 / 8 = 0.125 (i.e., a fraction of 1) as 12%.
(3) Emerson effect and Red drop: R. Emerson and C.M. Lewis (1943) observed that the quantum yield of photosynthesis decreases towards the far red end of the 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 quantum-yield 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.

$E=\frac{Quantum yield in combined beam–Quantum yield in red beam}{Quantum yield in far red beam}$

(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, P₇₀₀. Some physiologists also include carotenes and chlorophyll b in pigment system I. P₇₀₀ acts as the reaction centre. Thus, this system absorbs both wavelengths shorter and longer than 680nm.
(ii) Pigment system II or photosystem II: The main pigments of this system are chlorophyll a 673, P₆₈₀, chlorophyll b and phycobilins. This pigment system absorbs wavelengths shorter than 680nm only. P₆₈₀ acts as the reaction centre.
Pigment systems I and II are involved in non-cyclic electron transport, while pigment system I is involved only in cyclic electron transport. Photosystem I generates strong reductant NADPH. Photosystem II produces a strong oxidant that forms oxygen from water.
Comparison of photosystem I and photosystem II

(5) Photophosphorylation: Light phase includes the interaction of two pigment systems. PS I and PS II constitute various type of pigments. Arnon 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 I. Flow of electron is cyclic. If 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 I i.e., P₇₀₀. 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.

(ii) 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 NADPH₂. Here H₂O is utilized and O₂ evolution occurs. In this chain high energy electrons released from ‘P-680’ do not return to ‘P-680’ but pass through pheophytin, plastoquinone, cytochrome b₆–f complex, plastocyanin and then enter P-700. In this transfer of electrons from plastoquinone (PQ) to cytochrome b₆–f complex, ATP is synthesized. Because in this process high energy electrons released from ‘P-680’ do not return to ‘P-680’ and ATP (1 molecules) is formed, this is called Noncyclic photophosphorylation. ATP is synthesized at only one step.
This non-cyclic photophosphorylation is also known as Z-scheme (because of shape of path of electron-flow) and this was given by Hill and Bendall (1960). Non-cyclic photophosphorylation or Z-scheme is inhibited by CMU and DCMU.
(DCMU is a herbicide which kills the weed by inhibiting CO₂ fixation as it is a strong inhibitor of PS-II).

Pseudocyclic photophosphorylation: Arnon and his coworker (1954) demonstrated yet another kind of photophosphorylation. They observed that even in absence of CO₂ and NADP, if chlorophyll molecules are illuminated, it can produce ATP from ADP and iP (Inorganic phosphate) in presence of FMN or vit. K and oxygen. The process is thus very simple and requires no net chemical change for the formation of ATP and water. Arnon called this oxygen dependent FMN catalysed photophosphorylation or pseudocyclic photophosphorylation which involves the reduction of FMN with the production of oxygen. FMN is an auto-oxidisable hydrogen acceptor with the effect that the reduced FMN is reoxidised by oxygen. Thus the process can continue repeatedly to produce ATP.
Since this process can be continuously self repeated, it appears that a single molecule of water should be sufficient to operate pseudocyclic photophosphorylation to meet the requirement of ATP.

Dark phase: The pathway by which all photosynthetic eukaryotic organisms ultimately incorporate CO₂ into carbohydrate is known as carbon fixation or photosynthetic carbon reduction (PCR) cycle or dark reactions.
The dark reactions are sensitive to temperature changes, but are independent of light hence it is called dark reaction, however it depends upon the products of light reaction of photosynthesis, i.e., NADP .2H and ATP.
The carbon dioxide fixation takes place in the stroma of chloroplasts because it has enzymes essential for fixation of CO₂ and synthesis of sugar.
The techniques used for studying different steps were Radioactive tracer technique using ¹⁴C (Half life – 5720 years), Chromatography and Autoradiography and the material used was Chlorella (Cloacal alga) and Scenedesmus (these are microscopic, unicellular algae and can be easily maintained in laboratory).
The assimilation and reduction of CO₂ takes place in this reaction by which carbohydrate is synthesized through following three pathways:
(1) Calvin cycle: Calvin and Benson discovered the path of carbon in this process. This is known as C₃ cycle because CO₂ reduction is cyclic process and first stable product in this cycle is a 3-C compound (i.e., 3-Phosphoglyceric acid or 3-PGA).
Calvin cycle is divided into three distinct phases: Carboxylation, Glycolytic reversal, regeneration of RuBP.
In this cycle, CO₂ acceptor molecule is RuBP or RuDP (i.e., Ribulose 1, 5-biphosphate or Ribulose 1, 5-diphosphate). There occurs covalent bonding of CO₂ to RuBP and the enzyme catalyzing this reaction is RuBP-carboxylase/oxygenase (Rubisco).
As calvin cycle takes in only one carbon (as CO₂) at a time, so it takes six turns of the cycle to produce a net gain of six carbons (i.e., hexose or glucose).
In this cycle, for formation of one mole of hexose sugar (Glucose), 18 ATP and 12 NADPH₂ are used.
The plants in which this pathway of CO₂ reduction occurs, are called C-3 plants.
About 85% of plant species are C-3 plants, including cereals (e.g., barley, rice, oat, wheat), groundnut, sugarbeet, cotton, tobacco, spinach, soybean most trees and lawn grasses etc.

(2) Hatch and Slack cycle (C₄ cycle): Kortschak and Hart supplied CO₂ to the leaves of sugarcane, they found that the first stable product is a four carbon (C₄) compound oxalo acetic acid instead of 3-carbon atom compound. The detailed study of this cycle has introduced by M.D. Hatch and C.R. Slack (1966). So it is called as “Hatch and Slack cycle”. The stable product in C₄ plant is a dicarboxylic substance. Hence it is called dicarboxylic acid cycle or DCA-cycle. C₄ plants are true xerophytic plants.
The important C₄ plants are sugarcane, maize, Sorghum, Cyperus rotundus, Digitaria brownii, Amaranthus, etc. These plants have “Kranz” (German term meaning halo or wreath) type of leaf anatomy. The vascular bundles, in C₄ leaves are surrounded by a layer of bundle sheath cells that contain large number of chloroplasts. The chloroplasts in C₄ leaves are dimorphic (Two morphologically distinct types). The chloroplasts of bundle sheath cells are larger in size and arranged centripetally. They contain starch grains but lack grana. The mesophyll cells, on the other hand, contain normal types of chloroplasts. Mesophyll and bundle sheath cells are connected by plasmodesmata. The mesophyll cells perform C₄ cycle and the cells of bundle sheath perform C₃ cycle.

CO₂ taken from the atmosphere is accepted by phosphoenolpyruvic acid (PEP) present in the chloroplasts of mesophyll cells of these leaves, leading to the formation of a 4-C compound, oxaloacetic acid (OAA). This acid is converted to another 4-C acid, the malic acid which enters into the chloroplasts of bundle sheath cells and there undergoes oxidative decarboxylation yielding pyruvic acid (a 3-C compound) and CO₂. CO₂ released in bundle sheath cells reacts with Ribulose-1,5-biphosphate (RuBP) already present in the chloroplasts of bundle sheath cells and thus Calvin cycle starts from here. Pyruvic acid re-enters mesophyll cells and regenerates phosphoenol pyruvic acid. CO₂ after reacting with RuBP gives rise to sugars and other carbohydrates. In C₄ plants, there are 2 carboxylation reactions, first in mesophyll chloroplast and second in bundle sheath chloroplast.

C₄ plants are better photosynthesizers. There is no photorespiration in these plants. In C₄ plants, for formation of one molecule of hexose (glucose) 30 ATP and 12 NADPH₂ are required.
Characteristics of C₄ cycle
(1) C₄ species have greater rate of CO₂ assimilation than C₃ species. This is on account of the fact that
(i) PEP carboxylase has great affinity for CO₂.
(ii) C₄ plants show little photorespiration as compared to C₃ plants, resulting in higher production of dry matter.
(2) C₄ plants are more adapted to environmental stresses than C₃ plants.
(3) CO₂ fixation by C₄ plants requires more ATP than that by C₃ plants. This additional ATP is needed for conversion of pyruvic acid to phosphoenol pyruvic acid and its transport.
(4) CO₂ acceptor molecule in C₄ plants is PEP. Further, PEP-carboxylase (PEPCO) is the key enzyme (RuBP-carboxylase enzyme is negligible or absent in mesophyll chloroplast, but is present in bundle sheath chloroplast).
(3) Crassulacean acid metabolism (CAM): This dark CO₂ fixation pathway proposed by Ting (1971). It operates in succulent or fleshy plants e.g., Cactus, Sedum, Kalanchoe, Opuntia, Agave, Orchid, Pineapple and Bryophyllum helping them to continue photosynthesis under extremely dry condition.
The stomata of succulent plants remain closed during day and open during night to avoid water loss (Scotactive stomata). They store CO₂ during night in the form of malic acid in presence of enzyme PEP carboxylase. The CO₂ stored during night is used in Calvin cycle during day time. Succulents refix CO₂ released during respiration and use it during photosynthesis.
This diurnal change in acidity was first discovered in crassulacean plants e.g., Bryophyllum. So it is called as crassulacean acid metabolism.
Formation of malic acid during dark is called acidification or phase-I. Release of  for actual photosynthesis during day is called deacidification or phase-II.

Characteristics of CAM pathway
(1) There is decrease in pH during the night and increase in pH during the day.
(2) CAM plants have enzymes of both C₃ and C₄ cycle in mesophyll cells. This metabolism enable CAM plants to survive under xeric habitats. These plants have also the capability of fixing the CO₂ lost in respiration.
(3) Malic acid is stored in the vacuoles during the night which is decarboxylated to release  during the day.

5. PHOTORESPIRATION
Decker and Tio (1959) reported that light induces oxidation of photosynthetic intermediates with the help of oxygen in tobacco. It is called as photorespiration. The photorespiration is defined by Krotkov (1963) as an extra input of O₂ and extra release of CO₂ by green plants is light.
Photorespiration is the uptake of O₂ and release of CO₂ in light and results from the biosynthesis of glycolate in chloroplasts and subsequent metabolism of glycolate acid in the same leaf cell. Biochemical mechanism for photorespiration is also called glycolate metabolism.

Loss of energy occurs during this process. The process of photorespiration involves the involvement of chloroplasts, peroxisomes and mitochondria. RuBP carboxylase also catalyses another reaction which interferes with the successful functioning of Calvin cycle.
Biochemical mechanism
(1) Ribulose-1, 5-biphosphate  $\stackrel{{\mathrm{O}}_2}{\to }$ 2 Phosphoglycolic acid +3 Phosphoglyceric acid
(2) 2 Phosphoglycolic acid + H₂O  $\stackrel{\mathrm{Phosphatas} \mathrm{e}}{\to }$ Glycolic acid + Phosphoric acid.
(3) Glycolic acid + O₂ $\underset{\mathrm{Oxidase}}{\overset{\mathrm{Glycolic} \mathrm{acid}}{\to }}$ Glyoxylic acid + H₂O₂
         $2{\mathrm{H}}_2{\mathrm{O}}_2\stackrel{\mathrm{Catalase}}{\to }2{\mathrm{H}}_2\mathrm{O}+{\mathrm{O}}_2$
(4) Glyoxylic acid + Glutamic acid  $\underset{\mathrm{transamina} \mathrm{se}}{\overset{\mathrm{Glutamate} –\mathrm{glyoxylate}}{\to }}$  Glycine + α-keto glutaric acid
(5) 2 Glycine + H₂O + NAD⁺ →   Serine + CO₂ + NH₃ + NADH
(6) Serine + Glyoxylic acid → Hydroxypyruvic acid + Glycine Hydroxypyruvic acid → Glyceric acid
(7) Glyceric acid + ATP → 3 phosphoglyceric acid + ADP + phosphate
Importance of photorespiration
Photorespiration is quite different from respiration as no ATP or NADH are produced. Moreover, the process is harmful to plants because as much as half the photosynthetically fixed carbon dioxide (in the form of RuBP) may be lost into the atmosphere through this process.
Any increase in O₂ concentration would favour the uptake of O₂ rather than CO₂ and thus, inhibit photosynthesis for this rubisco functions as RuBP oxygenase. Photorespiration is closely related to CO₂ compensation point and occurs only in those plants which have high CO₂ compensation point such as C₃ plants.
Photorespiration generally occurs in temperate plants. Few photorespiring plants are: Rice, bean, wheat, barley etc. Inhibitors of glycolic acid oxidase such as hydroxy sulphonates inhibit the process of photorespiration. Unlike usual mitochondria respiration neither reduced coenzymes are generated in photorespiration nor the oxidation of glycolate is coupled with the formation of ATP molecules. Photorespiration (C₂ cycle) is enhanced by bright light, high temperature, high oxygen and low CO₂ concentration.

6. BACTERIAL PHOTOSYNTHESIS
Like green plants, some purple and green sulphur bacteria are capable of synthesizing their organic food in presence of light and in absence of O₂, which is known as bacterial photosynthesis.
Van Niel was the first to point out these similarities. Oxygen is not liberated in bacteria during process of photosynthesis. Their photosynthesis is non-oxygenic. Because bacteria use H₂S in place of water (H₂O) as hydrogen donor. Photosynthetic bacteria are anaerobic. Only one type of pigment system (PSI) is found in bacteria except cyanobacteria which possess both PSI and PSII. Bacteria has two type of photosynthetic pigments. Bacteriochlorophyll and Bacterioviridin.
The photosynthetic bacteria fall under three categories
(1) Green sulphur bacteria: It contains chlorobium chlorophyll, which absorb 720-750nm (far red light) of wavelength of light. e.g., Chlorobium.
(2) Purple sulphur bacteria: e.g., Chromatium.
(3) Purple non-sulphur bacteria: e.g., Rhodospirillum, Rhodopseudomonas.
Characteristics of bacterial photosynthesis are:
(1) No definite chloroplasts but contain simple structures having pigments called chromatophores (term coined by Schmitz).
(2) Contain chlorobium chlorophyll or bacterio-chlorophyll.
(3) Use longer wavelengths of light (720-950nm).
(4) No utilization of H₂O (but use H₂S or other reduced organic and inorganic substances).
(5) No evolution of O₂.
(6) Photoreductant is NADH₂ (Not NADPH₂).
(7) Only one photoact and hence one pigment system and thus one reaction centre, i.e., P₈₉₀.
(8) Cyclic photophosphorylation is dominant.
(9) It occurs in presence of light and in absence of O₂.

7. CHEMOSYNTHESIS
Some forms of bacteria obtain energy by chemosynthesis. This process of carbohydrate formation in which organisms use chemical reactions to obtain energy from inorganic compounds is called chemosynthesis. Such chemoautotrophic bacteria do not require light and synthesize all organic cell requirements from CO₂ and H₂O and salts at the expense of oxidation of inorganic substances like (H₂, NO₃^–, SO₄ or carbonate). Some examples of chemosynthesis are:
(1) Nitrifying bacteria: e.g., Nitrosomonas, Nitrosococcus, Nitrobacter etc.
(2) Sulphur bacteria: e.g., Beggiatoa, Thiothrix and Thiobacillus.
(3) Iron bacteria: e.g., Ferrobacillus, Leptothrix and Cladothrix.
(4) Hydrogen bacteria: e.g., Bacillus pentotrophus
(5) Carbon bacteria: e.g., Carboxydomonas, Bacillus oligocarbophilus.

8. FACTORS AFFECTING PHOTOSYNTHESIS
Blackmann’s law of limiting factors
F.F. Blackmann (1905) proposed the law of limiting factors according to which ‘when process is conditioned to its rapidity by a number of factors, the rate of process is limited by the pace of the slowest factor’. CO₂ is usually a limiting factor in photosynthesis under field conditions particularly on clear summer days under adequate water supply.
Blackmann’s law of limiting factor is modification of Liebig’s law of minimum, which states that rate of process controlled by several factors is only as rapid as the slowest factor permits. Theory of three cardinal points was given by Sachs in 1860. According to this concept, there is minimum, optimum and maximum for each factor. For every factor, there is a minimum value when photosynthesis starts, an optimum value showing highest rate and a maximum value, above which photosynthesis fails to take place.
Factors: The rate of photosynthetic process is affected by several external (Environmental) and internal factors.
External factors
(1) Light: The ultimate source of light for photosynthesis in green plants is solar radiation, which moves in the form of electromagnetic waves. Out of the total solar energy reaching to the earth about 2% is used in photosynthesis and about 10% is used in other metabolic activities. Light varies in intensity, quality (Wavelength) and duration. The effect of light on photosynthesis can be studied under these three headings.
(i) Light intensity: The total light perceived by a plant depends on its general form (viz., height, size of leaves, etc.) and arrangement of leaves. Of the total light falling on a leaf, about 80% is absorbed, 10% is reflected and 10% is transmitted.
In general, rate of photosynthesis is more in intense light than diffused light. (Up to 10% light is utilized in sugarcane, i.e., Most efficient converter).
Another photosynthetic superstar of field growing plants is Oenothera claviformis (Winter evening-primrose), which utilizes about 8% light.
However, this light intensity varies from plant to plant, e.g., more in heliophytes (sun loving plants) and less in sciophytes (shade loving plants). For a complete plant, rate of photosynthesis increases with increase in light intensity, except very high light intensity where ‘Solarization’ phenomenon occurs, i.e., photo-oxidation of different cellular components including chlorophyll occurs.
It also affects the opening and closing of stomata thereby affecting the gaseous exchange. The value of light saturation at which further increase is not accompanied by an increase in CO₂ uptake is called light saturation point.
(ii) Light quality: Photosynthetic pigments absorb visible part of the radiation i.e., 380mμ to 760mμ. For example, chlorophyll absorbs blue and red light. Usually plants show high rate of photosynthesis in the blue and red light. Maximum photosynthesis has been observed in red light than in blue light. The green light has minimum effect. On the other hand, red algae shows maximum photosynthesis in green light and brown algae in blue light.
(iii) Duration of light: Longer duration of light period favours photosynthesis. Generally, if the plants get 10 to 12hrs light per day it favours good photosynthesis. Plants can actively exhibit photosynthesis under continuous light without being damaged. Rate of photosynthesis is independent of duration of light.
(2) Temperature: The optimum temperature for photosynthesis is 20 to 35°C. If the temperature is increased too high, the rate of photosynthesis is also reduced by time factor which is due to denaturation of enzymes involved in the process. Photosynthesis occurs in some conifers at high altitudes at – 35°C. Some algae in hot springs can undergo photosynthesis even at 75°C.
(3) Carbon dioxide: Carbon dioxide present in the atmosphere is about 0.032% by volume and it is really a low concentration which acts as limiting factor in nature. If we increase the amount of CO₂ under laboratory conditions and if the light and temperature are not the limiting factors, the rate of photosynthesis increases. This increase is observed up to 1% of CO₂ concentration. At the same time very high concentration of CO₂ becomes toxic to plants and inhibits photosynthesis.
(4) Water: Water is an essential raw material in photosynthesis. This rarely, acts as a limiting factor because less than 1% of the water absorbed by a plant is used in photosynthesis. However, lowering of photosynthesis has been observed if the plants are inadequately supplied with water.
(5) Oxygen: Excess of O₂ may become inhibitory for the process. Enhanced supply of O₂ increases the rate of respiration simultaneously decreasing the rate of photosynthesis by the common intermediate substances. The concentration for oxygen in the atmosphere is about 21% by volume and it seldom fluctuates. O₂ is not a limiting factor of photosynthesis. An increase in oxygen concentration decreases photosynthesis and the phenomenon is called Warburg effect. (Reported by German scientist Warburg (1920) in Chlorella algae).
This is due to competitive inhibition of RuBP-carboxylase by increased O₂ levels, i.e., O₂ competes for active sites of RuBP-carboxylase enzyme with CO₂. The explanation of this problem lies in the phenomenon of photorespiration. If the amount of oxygen in the atmosphere decreases then photosynthesis will increase in C₃ cycle and no change in C₄ cycle.
(6) Pollutants and Inhibitors: The oxides of nitrogen and hydrocarbons present in smoke react to form peroxyacetyl nitrate (PAN) and ozone. PAN is known to inhibit Hill reaction. Diquat and Paraquat (Commonly called as Viologens) block the transfer of electrons between Q and PQ in PS. II. Other inhibitors of photosynthesis are monouron or CMU (Chlorophenyl dimethyl urea) diuron or DCMU (Dichlorophenyl dimethyl urea), bromocil and atrazine etc. Which have the same mechanism of action as that of viologens.
At low light intensities potassium cyanide appears to have no inhibiting effect on photosynthesis.
(7) Minerals: Presence of Mn⁺⁺ and Cl^– is essential for smooth operation of light reactions (Photolysis of water/evolution of oxygen) Mg⁺⁺, Cu⁺⁺ and Fe⁺⁺ ions are important for synthesis of chlorophyll.
Internal factors
(1) Protoplasmic factors: There is some unknown factor which affects the rate of photosynthesis.
These factors affect the dark reactions. The decline in the rate of photosynthesis at temperature above 30°C or at strong light intensities in many plants suggests the enzymatic nature of this unknown factor.
(2) Chlorophyll content: Chlorophyll is an essential internal factor for photosynthesis. The amount of CO₂ fixed by a gram of chlorophyll in an hour is called photosynthetic number or assimilation number. It is usually constant for a plant species but rarely it varies. The assimilation number of variegated variety of a species was found to be higher than the green leaves variety.
(3) Accumulation of end products: Accumulation of food in the chloroplasts reduces the rate of photosynthesis.
(4) Structure of leaves: The amount of CO₂ that reaches the chloroplast depends on structural features of the leaves like the size, position and behaviour of the stomata and the amount of intercellular spaces. Some other characters like thickness of cuticle, epidermis, presence of epidermal hairs, amount of mesophyll tissue, etc., influence the intensity and quality of light reaching in the chloroplast.