Photosynthesis in Higher Plants

Start with the definition the examiner wants. Photosynthesis is a physico-chemical process by which green plants use light energy to drive the synthesis of organic compounds. Two reasons make it the most important process on earth: it is the primary source of all food on the planet, and it is responsible for the release of oxygen into the atmosphere. Higher plants do it in the green parts, chiefly the leaves, inside the chloroplasts.

Two simple experiments from earlier classes are recalled in the chapter and can still be asked as 2-mark questions. In the variegated leaf experiment, a leaf with green and non-green patches is exposed to light and then tested for starch with iodine; only the green parts turn blue-black, proving that chlorophyll is needed. In the half-leaf experiment, part of a leaf is enclosed in a test tube containing potassium hydroxide soaked cotton (KOH absorbs CO₂) while the other half stays in the air. After exposure to light, the half exposed to air tests positive for starch and the half inside the tube does not, proving that CO₂ is needed. From these you already know that chlorophyll, light and CO₂ are all required.

The overall equation should be written the NCERT way, with twelve water molecules on the left, because the oxygen released comes from water and not from CO₂:

6CO₂ + 12H₂O → C₆H₁₂O₆ + 6H₂O + 6O₂

Read it as a redox equation: water is oxidised (it loses electrons and gives O₂), and CO₂ is reduced to carbohydrate. Everything that follows in the chapter is about how the light energy is captured (the light reaction) and how it is spent to reduce CO₂ (the dark reaction, better called the biosynthetic phase).

The chapter lists five scientists and the examiner expects you to match each name to its finding. Learn them as a story.

Joseph Priestley (1770) placed a burning candle in a closed bell jar; it went out. A mouse in a closed jar suffocated. When he put a mint plant in the jar with the candle or the mouse, the candle kept burning and the mouse stayed alive. He concluded that plants restore to the air whatever the burning candle or the breathing animal removes; he had found that plants release oxygen (he called the air 'dephlogisticated').

Jan Ingenhousz (1730 to 1799) repeated the experiment in light and in dark and showed that sunlight is essential to the process. Using an aquatic plant he saw that in bright sunlight small bubbles (oxygen) formed around the green parts, and in dark no bubbles formed. So only the green parts of the plant release oxygen, and only in light.

Julius von Sachs (1854) provided evidence that plants produce glucose when they grow and that it is usually stored as starch. He also showed that the green substance (chlorophyll) is located in special bodies in the cell, later called chloroplasts.

T. W. Engelmann (1843 to 1909) split light with a prism into its colours, illuminated a filament of the green alga Cladophora placed in a suspension of aerobic bacteria, and watched where the bacteria gathered. They accumulated mainly in the blue and red regions of the spectrum, where the most oxygen was being released. This gave the first action spectrum of photosynthesis, which matches the absorption spectrum of chlorophyll a and b.

Cornelius van Niel (1897 to 1985) worked with purple and green sulphur bacteria, which use H₂S instead of water and release sulphur, not oxygen. From this he proposed that photosynthesis is essentially a light-dependent reaction in which hydrogen from an oxidisable compound reduces CO₂ to carbohydrate, and that in green plants the hydrogen donor is water, so the O₂ released comes from water, not from CO₂. This was later proved using radioisotopes.

Photosynthesis takes place in the green parts of the plant, mainly in the mesophyll cells of the leaf, which have a large number of chloroplasts. Inside the mesophyll cell the chloroplasts align themselves along the cell walls so that they receive the optimum amount of incident light.

The structure to draw and label is NCERT Figure 11.1. A chloroplast has a double membrane envelope enclosing two compartments: the membranous system made of grana (stacks of thylakoids), the stroma lamellae that connect the grana, and the fluid stroma in which the membranes are embedded. Also mark the thylakoid lumen, the space inside a thylakoid, because it matters for chemiosmosis later.

The division of labour is a standard 2-mark answer. The membrane system (grana and stroma lamellae) is responsible for trapping light energy and for the synthesis of ATP and NADPH. That is the light reaction. In the stroma the enzymatic reactions take place that incorporate CO₂ into the plant to make sugar, which in turn forms starch. Those are the dark reactions. The name is a trap: the dark reactions do not require light directly, but they depend on the ATP and NADPH made by the light reaction, so in practice they run in daylight. NCERT points out that calling them dark reactions is misleading, and the better term is biosynthetic phase or carbon reactions.

Keep the two-compartment picture in mind for the whole chapter. Every light-driven step (water splitting, electron flow, proton pumping, ATP synthase) is a membrane event; every carbon-fixing step (RuBisCO, PEP carboxylase in mesophyll of C₄ plants, the Calvin cycle) is a stroma event.

Leaves look green because of a mixture of pigments, not one. Separate a leaf extract by paper chromatography and four bands appear: chlorophyll a (bright or blue-green in the chromatogram), chlorophyll b (yellow-green), xanthophylls (yellow) and carotenoids (yellow to yellow-orange). Pigments are substances that absorb specific wavelengths of light; the colour we see is what is not absorbed.

Two graphs are asked together (NCERT Figure 11.4). The absorption spectrum of a pigment is a plot of the amount of light absorbed against wavelength. The action spectrum of photosynthesis is a plot of the rate of photosynthesis (measured, for example, as O₂ evolved) against wavelength. Chlorophyll a shows peaks in the blue and the red regions and a trough in the green. The action spectrum has the same shape, with maximum photosynthesis in blue and red and least in green. Because the action spectrum overlaps the absorption spectrum of chlorophyll a so closely, chlorophyll a is the chief pigment associated with photosynthesis. The overlap is not perfect, and the small gap shows that other pigments also contribute.

Why keep chlorophyll b, xanthophylls and carotenoids at all? These are accessory pigments. They absorb light at wavelengths that chlorophyll a absorbs poorly and transfer the energy to chlorophyll a, so the plant uses a wider band of the incident light. They also protect chlorophyll a from photo-oxidation. That is the answer to NCERT exercise 5: a plant with chlorophyll b but no chlorophyll a would not photosynthesise, because only chlorophyll a at the reaction centre converts light energy into chemical energy; the accessory pigments can only hand energy over.

The band of wavelengths usable by plants, roughly 400 to 700 nm, is called photosynthetically active radiation (PAR). Green light (around 500 to 550 nm) is the least effective colour because it is largely reflected.

The light reaction, also called the photochemical phase, includes four events: light absorption, water splitting, oxygen release, and the formation of ATP and NADPH. All four happen on the thylakoid membranes.

The pigments are not scattered; they are organised into two discrete photosystems, Photosystem I (PS I) and Photosystem II (PS II), named in the order in which they were discovered, not the order in which they act. Each photosystem is a light harvesting complex (LHC) made of hundreds of pigment molecules bound to proteins. In each, a single chlorophyll a molecule forms the reaction centre, and all the other pigments (chlorophyll a, chlorophyll b, carotenoids) form the antenna, whose job is to absorb light of different wavelengths and funnel the energy to the reaction centre.

The two reaction centres differ in the wavelength at which their chlorophyll a absorbs best. In PS I the reaction centre chlorophyll a has its absorption peak at 700 nm and is called P700. In PS II the peak is at 680 nm and it is called P680. Draw NCERT Figure 11.5 when asked about the LHC: a fan of pigment molecules pointing inward to one reaction-centre chlorophyll a, with arrows showing energy transfer.

Why two photosystems? The energy needed to lift an electron all the way from water (a poor electron donor) to NADP⁺ (making the strong reductant NADPH) is more than one photon at a single centre can provide. So the electron is boosted twice, once at PS II and once at PS I. That is the logic of the Z scheme in the next section.

This is the diagram most often asked for 5 marks, NCERT Figure 11.6. Learn it as a sequence of six steps and draw it as a Z lying on its side, with redox potential on the vertical axis.

Step 1. In PS II the reaction centre chlorophyll a (P680) absorbs 680 nm red light and becomes excited. It gives up an electron to a primary electron acceptor, which now has a higher energy than the electron had in P680.

Step 2. The electron passes from the primary acceptor to an electron transport system (ETS) made of cytochromes. The electron moves downhill in terms of redox potential, and as it passes through the chain some of its energy is used to make ATP (see chemiosmosis, 11.6.3). The exemplar order of carriers is PS II, plastoquinone, cytochromes, PS I, ferredoxin.

Step 3. The electron reaches the pigments of PS I, filling the hole left when P700 loses its electron. In this way PS II continuously supplies electrons to PS I.

Step 4. P700 in PS I absorbs red light (700 nm), becomes excited, and passes an electron to another acceptor that has a higher redox potential.

Step 5. From this acceptor the electron again moves downhill, through ferredoxin, to NADP⁺. The enzyme NADP reductase on the stroma side adds two electrons and a proton to reduce NADP⁺ to NADPH + H⁺.

Step 6. PS II is left short of an electron. That hole is filled by electrons from water, which is split to give protons, electrons and O₂ (section 11.6.1).

This whole scheme of transfer of electrons, starting from PS II, uphill to the acceptor, downhill to PS I, excitation of electrons, transfer to another acceptor, and finally downhill to NADP⁺, is called the Z scheme, because of its shape when the carriers are placed on a redox-potential scale. The net products of one pass are ATP, NADPH and O₂. Because electrons flow in one direction from water to NADP⁺ and do not return, this is non-cyclic photophosphorylation.

P680, having lost its electron, must get one back or PS II stops. The electrons come from water. The water-splitting complex is associated with PS II, which itself is physically located on the inner side of the thylakoid membrane, facing the lumen. Water is split into protons (H⁺), electrons (e⁻) and oxygen (O₂). Write it as:

2H₂O → 4H⁺ + O₂ + 4e⁻

Three consequences are examined. First, the electrons replace those that PS II lost, so P680 is regenerated and the Z scheme keeps running. Second, the oxygen is a by-product that diffuses out; this is the source of atmospheric oxygen and the confirmation of van Niel's proposal that O₂ comes from water, not CO₂. Third, the protons are released inside the thylakoid lumen, which is one of the ways the proton gradient of chemiosmosis is built (section 11.6.3).

The splitting of water is also called photolysis of water, because light energy absorbed by PS II drives it. Manganese, calcium and chloride ions are associated with the water-splitting complex; NCERT does not go into the details, so a mention is enough. What NCERT does stress, and what MCQs test, is the location: water is split on the inner side of the thylakoid membrane, by the complex linked to PS II, and the O₂ released has its source in H₂O.

Photophosphorylation is the synthesis of ATP from ADP and inorganic phosphate in the presence of light, by living cells (chloroplasts). It is the light-driven counterpart of oxidative phosphorylation in mitochondria. There are two forms, and the comparison is one of the most regular 3-mark and 5-mark questions.

Non-cyclic photophosphorylation. Both PS II and PS I work in series. Electrons flow from water through PS II, the cytochrome chain, PS I and ferredoxin to NADP⁺, and do not return. Products: ATP and NADPH, with O₂ released from water. Site: the grana lamellae, whose membranes carry both photosystems.

Cyclic photophosphorylation. Only PS I is involved. The electron excited from P700 is passed to the acceptor but, instead of going to NADP⁺, is circulated back to the PS I complex through the electron transport chain. The cyclic flow makes only ATP; no NADPH is formed and no O₂ is released because water is not split. It possibly occurs in the stroma lamellae, since these membranes lack PS II and NADP reductase and therefore cannot run the non-cyclic route. Cyclic flow also takes over when only light of wavelengths beyond 680 nm is available, because such light excites P700 but not P680.

Why does the plant need a cyclic route at all? The Calvin cycle uses 3 ATP for every 2 NADPH, but non-cyclic flow makes roughly equal amounts. Cyclic photophosphorylation tops up the ATP without making extra NADPH.

For the tabular comparison, use these rows: photosystems involved (PS I and PS II versus PS I only), electron pathway (non-cyclic, from water to NADP⁺ versus cyclic, back to PS I), water splitting and O₂ evolution (yes versus no), products (ATP and NADPH versus ATP only), site (grana versus stroma lamellae), and light condition (both 680 and 700 nm versus beyond 680 nm).

How does electron flow make ATP? The answer is chemiosmosis, proposed by Peter Mitchell and used in both chloroplasts and mitochondria. ATP synthesis is linked to the development of a proton gradient across a membrane. In the chloroplast the protons accumulate inside the thylakoid lumen, while in mitochondria they accumulate in the intermembrane space. The gradient forms in three ways, each a marking point.

(1) Splitting of water takes place on the inner side of the membrane, so the protons it produces accumulate in the lumen.

(2) The primary electron acceptor of PS II, located towards the outer (stroma) side of the membrane, hands its electron not to an electron carrier but to an H carrier (plastoquinone). This molecule picks up a proton from the stroma as it takes the electron; when it passes the electron to the next carrier on the inner side of the membrane, it releases the proton into the lumen. So protons are pumped from stroma to lumen.

(3) NADP reductase sits on the stroma side of the membrane. The protons needed to reduce NADP⁺ to NADPH + H⁺ are taken from the stroma, lowering the proton concentration there further.

The result is a decrease of protons in the stroma and an accumulation in the lumen: a proton gradient and a pH difference across the thylakoid membrane. The gradient is broken down by movement of protons across the membrane through the transmembrane channel of the enzyme ATP synthase (ATPase). This enzyme has two parts. F₀ is embedded in the membrane and forms the channel that carries out facilitated diffusion of protons across it. F₁ protrudes on the outer surface of the membrane, on the side facing the stroma. The breakdown of the gradient releases enough energy to cause a conformational change in F₁, which makes the enzyme synthesise ATP from ADP and inorganic phosphate.

Summarise the whole light reaction in one sentence: it needs a membrane, a proton pump, a proton gradient and ATP synthase, and its products, ATP and NADPH, are used in the stroma to fix CO₂.

The products of the light reaction, ATP, NADPH and O₂, have different fates. O₂ diffuses out of the chloroplast. ATP and NADPH are used in the stroma to drive the processes that reduce CO₂ to sugars. This is the biosynthetic phase. It does not directly depend on light, but it depends on the products of the light reaction. If light stops, the biosynthetic phase continues for a short while and then stops; if light is restored, it restarts. That is the correct answer to 'is the term dark reaction justified'.

Historically, CO₂ fixation was traced by Melvin Calvin and co-workers using the radioactive isotope 14C in algal photosynthesis. They found that the first stable CO₂ fixation product is a 3-carbon organic acid, 3-phosphoglyceric acid (PGA). Plants in which this is the first product are called C₃ plants, and the pathway is the C₃ pathway or Calvin cycle. Later, in some plants, the first stable product was found to be a 4-carbon organic acid, oxaloacetic acid (OAA); these are C₄ plants.

The primary acceptor of CO₂ was a puzzle for a while because scientists expected a 2-carbon acceptor (2C + CO₂ = 3C). It turned out to be a 5-carbon ketose sugar, ribulose-1,5-bisphosphate (RuBP). RuBP + CO₂ gives an unstable 6-carbon compound that immediately splits into two molecules of 3-PGA. In C₄ plants the primary acceptor in the mesophyll is the 3-carbon phosphoenol pyruvate (PEP), giving the 4-carbon OAA.

One line to remember: the Calvin cycle occurs in all photosynthetic plants, whether they have the C₃ or the C₄ pathway. The C₄ route is an addition in front of the Calvin cycle, not a replacement.

The Calvin cycle was worked out by Calvin, Benson and Bassham. It runs in the stroma of every photosynthetic plant and has three stages: carboxylation, reduction and regeneration. Draw it as a circle (NCERT Figure 11.8) with CO₂ entering at the top, RuBP and 3-PGA on the ring, ATP and NADPH consumed on the reduction arm, ATP consumed on the regeneration arm, and sucrose (or triose phosphate) leaving.

Carboxylation. CO₂ is fixed into a stable organic intermediate. RuBP (5C) accepts CO₂ to give two molecules of 3-PGA. The enzyme is RuBP carboxylase, called RuBisCO because it also has an oxygenase activity (section 11.8). This is the most crucial step of the cycle.

Reduction. A series of reactions that lead to the formation of glucose. This stage uses 2 ATP for phosphorylation and 2 NADPH for reduction per CO₂ molecule fixed. The fixation of six CO₂ molecules and six turns of the cycle are needed to remove one molecule of glucose from the pathway.

Regeneration. RuBP, the CO₂ acceptor, must be regenerated for the cycle to continue. This needs 1 ATP per CO₂ for the phosphorylation that forms RuBP.

Now the bookkeeping that the 3-mark question tests. Per CO₂: 2 ATP + 1 ATP = 3 ATP and 2 NADPH. For one glucose (6 CO₂, six turns): 18 ATP and 12 NADPH. NCERT states it as a balance sheet: In: 6 CO₂, 18 ATP, 12 NADPH. Out: 1 glucose, 18 ADP, 12 NADP⁺. The ATP and NADPH are supplied by the light reaction, which is why the Calvin cycle cannot run for long in the dark.

Two connections make good answers. Six turns are needed because each turn fixes only one carbon and glucose has six. And the mismatch between 3 ATP and 2 NADPH per CO₂ explains why plants need cyclic photophosphorylation in addition to the non-cyclic route.

C₄ plants, mostly tropical grasses such as maize, sorghum and sugarcane, have oxaloacetic acid as the first CO₂ fixation product. They share four features that make them examination favourites: they have a special leaf anatomy, they tolerate higher temperatures, they respond to high light intensities, they lack photorespiration, and they have greater productivity of biomass.

Kranz anatomy. In a C₄ leaf the vascular bundles are surrounded by a ring of particularly large cells called bundle sheath cells; 'Kranz' means wreath, describing this arrangement. The bundle sheath cells may form several layers around the bundle. They have a large number of chloroplasts, thick walls impervious to gaseous exchange, and no intercellular spaces. C₃ leaves show no such sheath and the mesophyll is differentiated into palisade and spongy layers. Looking at a plant from outside you cannot tell C₃ from C₄; a transverse section of the leaf can.

The Hatch and Slack pathway (NCERT Figure 11.10) needs both cell types. In the mesophyll cells, the primary acceptor PEP (3C) fixes CO₂ to form OAA (4C), catalysed by PEP carboxylase (PEPcase). Mesophyll cells lack RuBisCO. OAA is converted to other 4-carbon acids such as malic acid or aspartic acid, which are transported to the bundle sheath cells. There the C₄ acid is decarboxylated, releasing CO₂ and a 3-carbon molecule (pyruvate). The bundle sheath cells are rich in RuBisCO but lack PEPcase, so the released CO₂ enters the Calvin cycle and sugar is made there. The 3-carbon molecule is transported back to the mesophyll, where it is converted to PEP again (this costs ATP), completing the cycle.

The purpose of all this is to be a CO₂ concentrating mechanism. By pumping carbon into the bundle sheath and releasing it there, the plant builds a high CO₂ concentration around RuBisCO, which keeps the enzyme working as a carboxylase and suppresses its oxygenase activity. That is why only a few cells (bundle sheath) run the Calvin cycle yet the plant is highly productive: those cells are never starved of CO₂, never lose fixed carbon to photorespiration, and the whole leaf feeds them.

RuBisCO is the most abundant enzyme in the world, and it has an odd feature: its active site can bind both CO₂ and O₂, which compete for it. The enzyme has a much greater affinity for CO₂ than for O₂, so which reaction wins depends on the relative concentration of the two gases at the enzyme.

In C₃ plants, on a hot bright day the stomata partly close, CO₂ inside the leaf falls and O₂ (a product of the light reaction) rises. Some O₂ now binds RuBisCO, and RuBP, instead of being carboxylated to two molecules of 3-PGA, is oxygenated to one molecule of phosphoglycerate (3C) and one molecule of phosphoglycolate (2C). This is photorespiration, a light-dependent, oxygen-consuming process. In the photorespiratory pathway there is no synthesis of sugars, no ATP and no NADPH; instead it results in the release of CO₂ with the utilisation of ATP. So it is a wasteful process that undoes part of the Calvin cycle's work. It involves the chloroplast, the peroxisome and the mitochondrion.

In C₄ plants photorespiration does not occur. They have the CO₂ concentrating mechanism of the Hatch and Slack pathway: C₄ acids made in the mesophyll are broken down in the bundle sheath cells, releasing CO₂ right where RuBisCO is, so the intracellular CO₂ concentration is high and the enzyme functions as a carboxylase, with oxygenase activity minimised. That is the full answer to NCERT exercise 4 (why RuBisCO carries out more carboxylation in C₄ plants) and to exemplar's 'why is photorespiration absent in C₄ plants'.

The consequence is a difference in productivity and tolerance. C₄ plants show high productivity, tolerate high temperatures, and can keep photosynthesising with partly closed stomata; C₃ plants lose up to a quarter of the carbon they fix on hot days to photorespiration. The exemplar also asks under what conditions C₄ plants are superior: high temperature, high light intensity and low CO₂ or water stress, precisely the conditions that favour the oxygenase activity of RuBisCO in C₃ plants.

The rate of photosynthesis depends on both plant factors (number, size, age and orientation of leaves, mesophyll cells and chloroplasts, internal CO₂ concentration and chlorophyll content) and external factors (sunlight, temperature, CO₂ and water). Because these act together, the chapter states Blackman's (1905) Law of Limiting Factors: 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. Learn this sentence; it is quoted verbatim in answers. Example: with plenty of light, CO₂ and chlorophyll but low temperature, the rate is set by temperature; raise the temperature and the rate rises until some other factor becomes limiting.

Light. Consider light quality, intensity and duration. At low intensities there is a linear relationship between incident light and CO₂ fixation. At higher intensities the rate does not keep rising, because other factors become limiting. Light saturation occurs at about 10 per cent of full sunlight, so except for plants in shade or in dense forests light is rarely a limiting factor in nature. Very high light causes breakdown of chlorophyll and a fall in the rate. NCERT Figure 11.11 shows the curve; in the rising region (A) light is limiting, at the plateau (C, D) some other factor is.

Carbon dioxide. CO₂ is the major limiting factor. Its concentration in the atmosphere is very low, between 0.03 and 0.04 per cent; raising it up to 0.05 per cent increases CO₂ fixation, but beyond that it is damaging over long periods. C₃ and C₄ plants respond differently. At low light both respond little; at high light both respond to higher CO₂. C₄ plants saturate at about 360 µl L⁻¹, while C₃ plants keep responding and saturate only beyond 450 µl L⁻¹, so present-day atmospheric CO₂ is limiting for C₃ plants. This is used in greenhouses: crops such as tomatoes and bell pepper are grown in CO₂-enriched air to raise yield.

Temperature. The dark reactions are enzymatic and therefore temperature controlled; light reactions are also temperature sensitive but to a much lesser extent. C₄ plants respond to higher temperatures and show a higher rate, while C₃ plants have a much lower temperature optimum. Tropical plants have a higher optimum than temperate plants, because plants adapt to their habitat.

Water. Water is a raw material, but its effect is felt more through the plant than through the reaction. Water stress closes the stomata, reducing CO₂ availability, and makes leaves wilt, reducing their surface area and metabolic activity.