Photosynthesis in Higher Plants

What Do We Know?

Photosynthesis is the process by which plants and some other organisms use sunlight to synthesise foods with the help of chlorophyll pigment. It is a vital process that sustains life on Earth by converting light energy into chemical energy in the form of organic compounds (sugars) and releasing oxygen.

From our basic understanding, we know a few things about photosynthesis:

It is the primary source of all food on Earth.
It is also responsible for the release of oxygen into the atmosphere.
It takes place in the green parts of plants, mainly the leaves.
Chlorophyll and light are essential for this process.

The basic equation representing photosynthesis is often shown as:

$ 6CO_2 + 6H_2O \xrightarrow{\text{Light, Chlorophyll}} C_6H_{12}O_6 + 6O_2 $

This simplified equation shows that carbon dioxide and water are converted into glucose (a sugar) and oxygen, using light energy captured by chlorophyll.

While this equation represents the overall process, photosynthesis is a complex series of reactions that occur in different stages within the plant cell. The subsequent sections will delve into the details of this process, the components involved, and the mechanisms by which it takes place.

Early Experiments

The understanding of photosynthesis has evolved over centuries through a series of experiments. Early scientists conducted experiments to figure out what was needed for plants to produce food.

Joseph Priestley (1770)

Priestley performed a series of experiments that revealed the essential role of air in the growth of green plants.
He observed that a candle burning in a closed space (bell jar) would extinguish, and a mouse would suffocate in the same space. This suggested that burning candles and breathing animals somehow damage the air.
He then placed a mint plant in the same bell jar where the candle had burned out. After a few days, he found that the air in the jar could now support the burning of a candle or the life of a mouse.
Conclusion: Plants restore the air what breathing animals and burning candles remove. He hypothesised that plants release a substance that purifies the air (later identified as oxygen).

Diagram illustrating Priestley's bell jar experiment with a candle, mouse, and mint plant

*(Image shows a bell jar experiment with a candle and mouse (air becomes 'bad'), and another bell jar with a mint plant restoring the air quality)*

Jan Ingenhousz (1779)

Ingenhousz built on Priestley's work and showed that sunlight is essential to the plant process that purifies air.
He conducted experiments with an aquatic plant (like Hydrilla). He observed that in bright sunlight, small bubbles were formed around the green parts of the plant, while in the dark, no bubbles were formed.
He identified these bubbles to be oxygen.
Conclusion: Green parts of plants release oxygen only in the presence of sunlight.

Julius von Sachs (1854)

Sachs provided evidence that glucose is produced when plants grow, and that glucose is usually stored as starch.
He showed that the green substance in plants (chlorophyll) is located in special bodies within plant cells (later called chloroplasts).
He found that glucose is made in the green parts of the plant and is stored as starch, particularly in the leaves.

T.W. Engelmann (1883)

Engelmann performed a key experiment using a prism to split light into its spectral components (VIBGYOR).
He placed a filamentous alga (e.g., Spirogyra) in a suspension of aerobic bacteria and illuminated it with the spectrum of light.
He observed that the bacteria accumulated mainly in the regions of blue and red light of the split spectrum.
Conclusion: The bacteria accumulated where oxygen was being produced by the alga, indicating that photosynthesis was occurring maximally in the blue and red regions of the spectrum. This provided the first action spectrum of photosynthesis.

Diagram illustrating Engelmann's experiment with Spirogyra, aerobic bacteria, and a prism

*(Image shows a setup with a prism splitting light, a slide with filamentous alga and bacteria, showing bacteria clustered in the blue and red light regions)*

Cornelius van Niel (1930s)

Van Niel, studying purple and green bacteria (which use $H_2S$ instead of $H_2O$ as the hydrogen donor), made a significant contribution.
He showed that photosynthesis is essentially a light-dependent reaction in which hydrogen from a suitable oxidisable compound reduces carbon dioxide to carbohydrates.
In green plants, $H_2O$ is the hydrogen donor, and it is oxidised to oxygen ($O_2$).
$ CO_2 + H_2O \xrightarrow{\text{Light}} (CH_2O) + O_2 $
In purple and green bacteria, $H_2S$ is the hydrogen donor, and it is oxidised to sulphur ($S$) or sulphate ($SO_4^{2-}$).
$ CO_2 + H_2S \xrightarrow{\text{Light}} (CH_2O) + 2S $
Conclusion: The oxygen evolved during photosynthesis by green plants comes from water ($H_2O$), not from carbon dioxide ($CO_2$). This was later confirmed by using radioisotopes of oxygen ($^{18}O$) in water.

$ 6CO_2 + 12H_2^{18}O \xrightarrow{\text{Light}} C_6H_{12}O_6 + 6H_2O + 6^{18}O_2 $

$ 6C^{18}O_2 + 12H_2O \xrightarrow{\text{Light}} C_6H_{12}^{18}O_6 + 6H_2^{18}O + 6O_2 $ (This did not happen)

These early experiments laid the foundation for our understanding of the basic requirements and processes of photosynthesis.

Where Does Photosynthesis Take Place?

Photosynthesis takes place in the green parts of plants, specifically in the chloroplasts.

Chloroplasts: The Site of Photosynthesis

Chloroplasts are double membrane-bound organelles found mainly in the mesophyll cells of leaves.
Within the chloroplast, there are two main regions:
- Grana: Stacks of flattened membranous sacs called thylakoids. Thylakoid membranes contain chlorophyll and other pigments and are the site of the Light-dependent reactions.
- Stroma: The fluid-filled space surrounding the grana. It contains enzymes required for the synthesis of sugars, as well as chloroplast DNA and ribosomes. The stroma is the site of the Light-independent reactions (Calvin cycle).
Thylakoids in different grana are connected by flat membranous tubules called stromal lamellae.

Diagram showing the ultrastructure of a chloroplast highlighting outer/inner membrane, stroma, grana (stacks of thylakoids), and stromal lamellae

*(Image shows a cross-section of a chloroplast illustrating its main components)*

Two Stages of Photosynthesis:

Photosynthesis is divided into two main stages:

Light-dependent reactions (Light reactions): These reactions occur in the thylakoid membranes. They require light energy to produce ATP (chemical energy) and NADPH (reducing power). Water is split, releasing oxygen.
Light-independent reactions (Dark reactions / Calvin cycle): These reactions occur in the stroma. They do not directly require light, but they depend on the ATP and NADPH produced during the light reactions to fix carbon dioxide ($CO_2$) and synthesise sugars.

The light reactions and the dark reactions are interconnected. The products of the light reactions (ATP and NADPH) are used to drive the dark reactions, and the products of the dark reactions (ADP and NADP$^+$) are recycled back to the light reactions.

How Many Types Of Pigments Are Involved In Photosynthesis?

Photosynthesis relies on pigments that absorb light energy. These pigments are primarily located in the thylakoid membranes of chloroplasts.

Photosynthetic Pigments

The main photosynthetic pigments in higher plants are:

Chlorophylls: The primary photosynthetic pigments. They absorb light mainly in the blue and red regions of the spectrum and reflect green light (hence plants appear green).
- Chlorophyll a: The main pigment, the primary reaction centre of photosynthesis. It absorbs light most strongly in the blue-violet and red regions.
- Chlorophyll b: An accessory pigment. It absorbs light in regions slightly different from chlorophyll a and transfers the energy to chlorophyll a.
Carotenoids: Accessory pigments (yellow, orange, or red pigments).
- Carotenes: (e.g., $\beta$-carotene).
- Xanthophylls: (e.g., lutein).
Functions of carotenoids:
- Absorb light in regions of the spectrum where chlorophylls do not absorb effectively (e.g., green-yellow region), thus broadening the range of light used for photosynthesis.
- Protect chlorophyll from photo-oxidation (damage by excess light energy).

Other pigments like phycobilins are found in algae and cyanobacteria, but not in higher plants.

Absorption Spectra and Action Spectra

Absorption spectrum: A graph showing the relative absorption of light by a pigment at different wavelengths. Chlorophyll a and chlorophyll b have distinct absorption peaks in the blue and red regions. Carotenoids absorb in the blue-green region.
Action spectrum: A graph showing the rate of photosynthesis at different wavelengths of light. It shows that photosynthesis is maximal in the blue and red regions of the spectrum.

The action spectrum of photosynthesis closely matches the absorption spectrum of chlorophyll a, but it is slightly broader due to the contribution of accessory pigments (chlorophyll b and carotenoids) which absorb light and transfer the energy to chlorophyll a.

Graph showing absorption spectra of chlorophyll a, chlorophyll b, and carotenoids, and the action spectrum of photosynthesis

*(Image shows overlapping curves: absorption spectra of chlorophyll a, b, and carotenoids plotted against wavelength, and the action spectrum of photosynthesis plotted against wavelength, showing peaks in blue and red regions)*

Photosystems

The photosynthetic pigments are organised into two discrete photosystems (or pigment systems) located in the thylakoid membranes. Each photosystem consists of a reaction centre and associated light-harvesting complexes (antennae).

Reaction Centre: Contains a special molecule of chlorophyll a that absorbs light energy and converts it into chemical energy by initiating electron transfer.
Light-harvesting complexes (Antennae): Consist of hundreds of pigment molecules (chlorophyll b and carotenoids) bound to proteins. These pigments absorb light energy at different wavelengths and transfer this energy to the reaction centre through resonance energy transfer.

There are two photosystems:

Photosystem I (PS I): The reaction centre chlorophyll a has an absorption peak at 700 nm, hence it is called P700. PS I is found in both grana and stromal lamellae.
Photosystem II (PS II): The reaction centre chlorophyll a has an absorption peak at 680 nm, hence it is called P680. PS II is mainly found in the grana thylakoids.

The two photosystems work together to carry out the light-dependent reactions.

What Is Light Reaction?

The Light-dependent reactions (Light reactions) are the first stage of photosynthesis. They occur in the thylakoid membranes and directly utilise light energy to produce ATP and NADPH, and release oxygen as a byproduct.

Processes in Light Reactions:

Light absorption: Photosynthetic pigments (chlorophylls, carotenoids) absorb light energy.
Water splitting: Water molecules are split, releasing oxygen, protons ($H^+$), and electrons.
Light energy conversion: Light energy is converted into chemical energy in the form of ATP and NADPH. This involves electron transport and proton gradients.

Electron Transport Chain

The synthesis of ATP and NADPH is coupled to the movement of electrons through a series of electron carriers located in the thylakoid membrane. This is called the Electron Transport Chain (ETC).

The electrons follow two main pathways:

Non-cyclic photophosphorylation (Z-scheme): Involves both PS II and PS I. This process produces both ATP and NADPH, and releases oxygen.
Cyclic photophosphorylation: Involves only PS I. This process produces only ATP.

Summary Products of Light Reaction:

ATP (Adenosine Triphosphate) - Chemical energy currency.
NADPH (Nicotinamide Adenine Dinucleotide Phosphate, reduced form) - Reducing power.
Oxygen ($O_2$) - Released as a byproduct from the splitting of water.

These products (ATP and NADPH) are then used in the light-independent reactions (Calvin cycle) to fix carbon dioxide and synthesise sugars.

The Electron Transport

The electron transport chain in the thylakoid membrane is a series of redox reactions where electrons are transferred from one carrier molecule to the next, releasing energy at certain steps. This energy is used to produce ATP and NADPH.

Splitting Of Water

Water splitting (or photolysis of water) is an essential part of the non-cyclic electron transport chain. This process occurs on the inner side of the thylakoid membrane and is associated with PS II.

Reaction:

$ 2H_2O \rightarrow 4H^+ + O_2 + 4e^- $

Function of water splitting:

Provides electrons to replace the electrons lost by the reaction centre of PS II (P680).
Releases protons ($H^+$) into the lumen (space inside the thylakoid), contributing to the proton gradient.
Releases oxygen ($O_2$) as a gas, which is then released into the atmosphere.

This reaction requires enzymes and is catalysed by a complex associated with PS II, including Manganese (Mn), Calcium (Ca), and Chlorine (Cl) ions.

Cyclic And Non-Cyclic Photo-Phosphorylation

Photophosphorylation is the synthesis of ATP from ADP and inorganic phosphate (Pi) using light energy. It occurs in chloroplasts. There are two types:

Non-cyclic Photophosphorylation (Z-scheme):

Involves both PS II and PS I, connected by an electron transport chain.
Electrons follow a non-cyclic path. They are donated by water and ultimately accepted by NADP$^+$.
Steps:
1. PS II (P680) absorbs light. Electron is excited and leaves P680.
2. P680 becomes oxidised (P680$^+$), a strong oxidant, which accepts electrons from water splitting.
3. The excited electron from P680 is transferred to a primary electron acceptor.
4. From the primary acceptor, the electron moves downhill through an electron transport chain (including plastoquinone, cytochrome b6f complex, plastocyanin) to PS I. This electron transport is coupled to the pumping of protons from the stroma into the thylakoid lumen, creating a proton gradient for ATP synthesis.
5. PS I (P700) absorbs light. Electron is excited and leaves P700.
6. P700 becomes oxidised (P700$^+$), which accepts the electron from plastocyanin (from the PS II pathway).
7. The excited electron from P700 is transferred to another primary electron acceptor, and then moves through carriers (including ferredoxin) to NADP$^+$.
8. NADP$^+$ accepts the electron (and a proton from the stroma) and gets reduced to NADPH + $H^+$ by the enzyme NADP reductase (located on the stroma side of the thylakoid membrane).
Products: ATP, NADPH, and $O_2$ (from water splitting).
This process creates a potential difference across the thylakoid membrane, which drives ATP synthesis via chemiosmosis.

Diagram illustrating the Z-scheme (non-cyclic electron transport) in the thylakoid membrane

*(Image shows a diagram depicting the Z-scheme: PS II absorbing light, electron flow to primary acceptor, down through ETC to PS I (pumping protons), PS I absorbing light, electron flow to NADP+, reducing it to NADPH. Shows water splitting releasing O2 and electrons for PS II. Indicates ATP synthesis linked to proton gradient)*

Cyclic Photophosphorylation:

Involves only PS I.
The electron released by P700 is cycled back to P700 through an electron transport chain (including ferredoxin, cytochrome b6f complex, plastocyanin).
This cyclic flow is also coupled to proton pumping across the thylakoid membrane, generating a proton gradient.
Products: Only ATP. NADPH and $O_2$ are not produced (as water splitting is not involved, and electrons are not transferred to NADP$^+$).
Occurs in the stromal lamellae (which lack PS II and NADP reductase).
Cyclic photophosphorylation might occur when only light of wavelength 700 nm is available, or when NADPH is already accumulated, or when more ATP is required compared to NADPH.

Diagram illustrating cyclic electron transport involving only PS I

*(Image shows a simplified cycle diagram: PS I absorbing light, electron flowing to primary acceptor, then cyclically back to PS I via an ETC (including cytochrome b6f), indicating proton pumping for ATP synthesis)*

Chemiosmotic Hypothesis

The Chemiosmotic Hypothesis, proposed by Peter Mitchell, explains how ATP is synthesised in chloroplasts (and mitochondria) using the energy of proton gradients.

Steps in Chemiosmotic ATP Synthesis in Chloroplasts:

1. Proton Gradient Formation: A proton gradient is established across the thylakoid membrane, with a higher concentration of protons ($H^+$) in the thylakoid lumen and a lower concentration in the stroma. This gradient is created by:

Water splitting: Occurs on the inner side of the thylakoid membrane, releasing protons into the lumen.
Electron transport: As electrons move through the electron transport chain, particularly the cytochrome b6f complex, protons are pumped from the stroma into the lumen.
NADP$^+$ reduction: NADP$^+$ reductase is on the stroma side. Protons from the stroma are consumed to reduce NADP$^+$ to NADPH.

This creates both a proton concentration gradient and an electrical potential difference (lumen positive, stroma negative) across the thylakoid membrane.

2. Proton Flow through ATP Synthase: The thylakoid membrane contains an ATP synthase enzyme complex, which has two parts:

CF$_0$: An integral membrane protein channel that spans the membrane and provides a pathway for protons to diffuse across the membrane from the lumen to the stroma (down their electrochemical gradient).
CF$_1$: A peripheral membrane protein located on the stroma side of the membrane.

3. ATP Synthesis: The energy released by the movement of protons through the CF$_0$ channel causes a conformational change in the CF$_1$ part, which catalyses the synthesis of ATP from ADP and Pi on the stroma side.

$ ADP + Pi \xrightarrow{\text{ATP Synthase}} ATP $

Diagram illustrating the chemiosmotic hypothesis for ATP synthesis in a thylakoid membrane

*(Image shows a thylakoid membrane illustrating proton ($H^+$) accumulation in the lumen, electron transport chain components pumping protons, NADP+ reduction using stromal protons, and ATP synthase (CF0-CF1) allowing proton flow from lumen to stroma to generate ATP)*

Chemiosmosis is the mechanism by which light energy, through electron transport, is coupled to the synthesis of ATP. The ATP and NADPH produced during the light reactions are released into the stroma, where they are available for the next stage of photosynthesis.

Where Are The Atp And Nadph Used?

The ATP and NADPH produced during the light-dependent reactions are used to drive the synthesis of sugars from carbon dioxide. This process constitutes the Light-independent reactions, also known as the Calvin cycle or the C3 pathway.

These reactions occur in the stroma of the chloroplasts. They are 'light-independent' because they do not directly require light, but they are dependent on the energy (ATP) and reducing power (NADPH) generated by the light-dependent reactions.

The Primary Acceptor Of Co2

In the Calvin cycle, carbon dioxide ($CO_2$) is first incorporated into an organic molecule. The molecule that initially accepts $CO_2$ is crucial for this process.

Discovery of the Primary CO$_2$ Acceptor:

In the 1940s and 1950s, Melvin Calvin and his colleagues used radioactive $^{14}C$ to trace the path of carbon in photosynthesis (Calvin-Benson cycle).
They found that the first stable organic product formed after $CO_2$ fixation was a 3-carbon compound, 3-Phosphoglyceric Acid (3-PGA).
Since the first product was a 3-carbon compound, they initially thought the $CO_2$ acceptor was a 2-carbon compound. However, further research revealed that the $CO_2$ acceptor is actually a 5-carbon sugar.
The primary $CO_2$ acceptor in the Calvin cycle is a 5-carbon ribonucleotide diphosphate called Ribulose-1,5-bisphosphate (RuBP).

The enzyme responsible for this initial fixation of $CO_2$ to RuBP is RuBP carboxylase/oxygenase, commonly known as RuBisCO. RuBisCO is the most abundant enzyme in the world.

This reaction, where $CO_2$ is incorporated into RuBP, is called carboxylation.

The Calvin Cycle

The Calvin cycle is the metabolic pathway where $CO_2$ is fixed and reduced to form carbohydrates using ATP and NADPH. It has three main stages:

Carboxylation: $CO_2$ is fixed by RuBisCO to RuBP, forming an unstable 6-carbon intermediate, which immediately breaks down into two molecules of the 3-carbon compound, 3-PGA.
$ RuBP \:(5C) + CO_2 \xrightarrow{\text{RuBisCO}} \text{Unstable 6C intermediate} \rightarrow 2 \times \text{3-PGA} \:(3C) $

This is the first stable product of the Calvin cycle, making it the C3 pathway.

Reduction: The 3-PGA molecules are converted into 3-carbon sugars (Glyceraldehyde-3-phosphate). This stage requires ATP and NADPH from the light reactions.
- 3-PGA is phosphorylated by ATP to form 1,3-bisphosphoglycerate.
- 1,3-bisphosphoglycerate is reduced by NADPH to form glyceraldehyde-3-phosphate (a triose phosphate).
- For every molecule of $CO_2$ fixed, 2 molecules of 3-PGA are formed, requiring 2 ATP and 2 NADPH for reduction to triose phosphate.
- Glyceraldehyde-3-phosphate can be converted into glucose, sucrose, or starch.

Regeneration: RuBP, the $CO_2$ acceptor molecule, is regenerated from the remaining triose phosphate molecules. This stage requires ATP.
- For every one molecule of $CO_2$ fixed, only 1 molecule of triose phosphate can be exported from the cycle to form sugars. The remaining triose phosphate is used to regenerate RuBP.
- To regenerate 1 molecule of RuBP, 5 molecules of triose phosphate are needed (as 5 triose phosphates contain $5 \times 3C = 15C$, equal to the carbon in 3 RuBP molecules).
- This regeneration requires 1 ATP molecule per RuBP regenerated.

Diagram illustrating the Calvin Cycle (C3 pathway) with carboxylation, reduction, and regeneration stages

*(Image shows a cycle diagram illustrating the Calvin cycle: CO2 combining with RuBP (carboxylation), forming 3-PGA, reduction using ATP and NADPH to form triose phosphates, and regeneration of RuBP using ATP, showing carbon flow and ATP/NADPH usage)*

Inputs and Outputs of the Calvin Cycle:

To produce one molecule of glucose (a 6-carbon sugar), the Calvin cycle needs to fix 6 molecules of $CO_2$. This requires the cycle to turn 6 times.

For every 6 $CO_2$ molecules fixed (to produce 1 glucose):

ATP consumed: $6 \times 3 = 18$ ATP (3 per $CO_2$: 2 in reduction + 1 in regeneration)
NADPH consumed: $6 \times 2 = 12$ NADPH (2 per $CO_2$: in reduction)
Output: 1 molecule of Glucose (or equivalent hexose).

The overall equation for the Calvin cycle using the products of light reaction:

$ 6CO_2 + 18 ATP + 12 NADPH + 12 H_2O \rightarrow C_6H_{12}O_6 + 18 ADP + 18 Pi + 12 NADP^+ + 6 H_2O $

Simplified net equation:

$ 6CO_2 + 18 ATP + 12 NADPH \rightarrow C_6H_{12}O_6 + 18 ADP + 18 Pi + 12 NADP^+ $

Example 1. How many ATP and NADPH molecules are required to produce 3 molecules of glucose through the Calvin cycle?

Answer:

To produce 1 molecule of glucose, the Calvin cycle requires 18 ATP and 12 NADPH molecules.

To produce 3 molecules of glucose, the requirement will be $3 \times$ the requirement for 1 glucose molecule.

ATP required = $3 \times 18 = 54$ ATP molecules.

NADPH required = $3 \times 12 = 36$ NADPH molecules.

So, 54 ATP and 36 NADPH molecules are required to produce 3 molecules of glucose.

The C4 Pathway

While the Calvin cycle (C3 pathway) is common to all photosynthetic plants, some plants, particularly those adapted to dry tropical regions, have evolved an alternative pathway for carbon dioxide fixation called the C4 pathway or Hatch-Slack pathway.

Significance of C4 Pathway:

C4 plants are adapted to conditions of high temperature, high light intensity, low $CO_2$ concentration, and water stress.
They have a mechanism to increase the concentration of $CO_2$ at the site of the Calvin cycle, thus minimising photorespiration (a wasteful process, discussed later).
They are often more efficient in photosynthesis at high light intensities and do not show photorespiration.

Kranz Anatomy:

C4 plants exhibit a special type of leaf anatomy called Kranz anatomy (Kranz means 'wreath' in German). This anatomy is characterised by:

Presence of large bundle sheath cells surrounding the vascular bundles. These cells are arranged in a wreath-like manner.
Bundle sheath cells have thick walls, are impermeable to gaseous exchange, and have numerous chloroplasts.
Mesophyll cells are scattered, as in C3 plants, but are closely associated with bundle sheath cells.
The vascular bundles are surrounded by mesophyll cells, which are further surrounded by bundle sheath cells.

Diagram showing Kranz anatomy in a C4 leaf cross-section

*(Image shows a cross-section of a C4 leaf vein area, illustrating the vascular bundle surrounded by large bundle sheath cells (with chloroplasts) which are then surrounded by mesophyll cells)*

The C4 Pathway Steps:

The C4 pathway involves two types of photosynthetic cells: mesophyll cells and bundle sheath cells. $CO_2$ is fixed twice.

1. Initial $CO_2$ fixation in Mesophyll cells:

In the cytoplasm of mesophyll cells, $CO_2$ is accepted by a 3-carbon compound, Phosphoenolpyruvate (PEP).
This reaction is catalysed by the enzyme PEP carboxylase or PEPcase. This enzyme has a high affinity for $CO_2$ and does not bind to $O_2$.
The product is a 4-carbon organic acid, usually Oxaloacetic Acid (OAA).
$ PEP \:(3C) + CO_2 \xrightarrow{\text{PEPcase}} OAA \:(4C) $
OAA is quickly converted into other 4-carbon organic acids, such as malate or aspartate, which are transported from the mesophyll cells into the adjacent bundle sheath cells via plasmodesmata.

2. Decarboxylation in Bundle Sheath cells:

In the bundle sheath cells, the 4-carbon acids (malate or aspartate) are broken down (decarboxylated) to release $CO_2$ and a 3-carbon compound (pyruvate).
This released $CO_2$ is then fixed by RuBisCO into RuBP, entering the Calvin cycle (C3 cycle) in the bundle sheath cells.
The concentration of $CO_2$ in the bundle sheath cells becomes high, which saturates RuBisCO and suppresses photorespiration.

3. Regeneration of PEP in Mesophyll cells:

The 3-carbon compound (pyruvate) is transported back to the mesophyll cells.
In the mesophyll cells, pyruvate is phosphorylated using ATP to regenerate PEP, which is ready to accept another molecule of $CO_2$.
$ Pyruvate \:(3C) + ATP \rightarrow PEP \:(3C) + AMP + Pi $

Diagram illustrating the C4 pathway involving mesophyll and bundle sheath cells

*(Image shows a diagram illustrating the C4 pathway across mesophyll and bundle sheath cells: CO2 entering mesophyll, fixation by PEPcase to OAA/malate, transport to bundle sheath, decarboxylation releasing CO2, CO2 entering Calvin cycle in bundle sheath, pyruvate returning to mesophyll, regeneration of PEP using ATP)*

Energy Requirement of C4 Pathway:

For every molecule of $CO_2$ fixed, the C4 pathway requires more ATP than the C3 pathway because of the extra step of PEP regeneration.

C3 cycle requires 3 ATP and 2 NADPH per $CO_2$ fixed.
C4 pathway requires an additional 2 ATP (for regenerating PEP from pyruvate) per $CO_2$ fixed in the mesophyll cell.

Total energy requirement for C4 pathway: (3 ATP + 2 NADPH) in bundle sheath C3 cycle + 2 ATP in mesophyll = 5 ATP and 2 NADPH per $CO_2$ fixed.

Although C4 plants require more ATP, their ability to concentrate $CO_2$ minimises photorespiration, which saves energy that would otherwise be lost in C3 plants, making them more efficient in certain environments.

Examples of C4 Plants:

Maize ($Zea \: mays$)
Sugarcane ($Saccharum \: officinarum$)
Sorghum ($Sorghum \: bicolor$)
Amaranthus ($Amaranthus$)

Feature	C3 Plants	C4 Plants
Primary CO$_2$ acceptor	RuBP (5C)	PEP (3C)
Primary CO$_2$ fixing enzyme	RuBisCO	PEPcase
First stable product	3-PGA (3C)	OAA (4C)
Site of Calvin cycle	Mesophyll cells	Bundle sheath cells
Leaf anatomy	Typical (no Kranz)	Kranz anatomy (Bundle sheath cells)
Photorespiration	High (especially in high O$_2$, low CO$_2$)	Negligible/Absent
Efficiency at high light intensity	Lower (saturates at lower light)	Higher (no saturation)
Efficiency at low CO$_2$ concentration	Lower	Higher (PEPcase has high affinity)
ATP and NADPH per CO$_2$	3 ATP, 2 NADPH	5 ATP, 2 NADPH
Adaptation	Temperate regions	Dry tropical regions (high temp, light)
Examples	Rice, Wheat, Potato	Maize, Sugarcane, Sorghum

Photorespiration

Photorespiration is a wasteful process that occurs in C3 plants. It is a light-dependent process that consumes oxygen and releases carbon dioxide, without producing ATP or NADPH, and reducing the efficiency of photosynthesis.

Mechanism of Photorespiration:

The enzyme RuBisCO, which catalyses the carboxylation of RuBP in the Calvin cycle, also has oxygenase activity.
In the presence of high oxygen concentration relative to carbon dioxide, RuBisCO binds to $O_2$ instead of $CO_2$.
This leads to the oxidation of RuBP.
$ RuBP \:(5C) + O_2 \xrightarrow{\text{RuBisCO}} 3-PGA \:(3C) + Phosphoglycolate \:(2C) $
Phosphoglycolate (2C) is then metabolised through a series of reactions involving three organelles: chloroplasts, peroxisomes, and mitochondria. This process consumes oxygen and ATP, and releases $CO_2$.

The net result of photorespiration is the loss of fixed carbon as $CO_2$ and no energy production (ATP or NADPH). It effectively reduces the efficiency of photosynthesis.

Diagram illustrating the photorespiration pathway involving chloroplast, peroxisome, and mitochondrion

*(Image shows a diagram illustrating the flow of carbon compounds (RuBP, Phosphoglycolate, Glycolate, Glycine, Serine, 3-PGA) through chloroplast, peroxisome, and mitochondrion, showing O2 uptake and CO2 release)*

Factors Favouring Photorespiration:

High oxygen concentration
Low carbon dioxide concentration
High temperature
High light intensity

These conditions often occur when stomata are partially closed during hot, dry weather to conserve water. This limits $CO_2$ uptake, while $O_2$ produced during photosynthesis builds up inside the leaf.

Why Photorespiration is Absent or Negligible in C4 Plants:

C4 plants have Kranz anatomy and the C4 pathway, which effectively concentrates $CO_2$ in the bundle sheath cells (site of the Calvin cycle).
The high $CO_2$ concentration around RuBisCO in bundle sheath cells minimises the oxygenase activity of RuBisCO and favours its carboxylation activity.
Also, PEPcase in mesophyll cells does not bind oxygen, so the initial $CO_2$ fixation is not affected by high $O_2$.
Thus, C4 plants avoid the wasteful process of photorespiration and are more efficient in carbon fixation under conditions that favour photorespiration in C3 plants.

Factors Affecting Photosynthesis

The rate of photosynthesis is influenced by several factors, both internal and external. Understanding these factors is important for optimising plant growth and yield.

Law of Limiting Factors (Blackman's Law, 1905)

This law states that if a process is conditioned as to its rapidity by a number of separate factors, the rate of the process is limited by the pace of the slowest factor.

In photosynthesis, this means that at any given time, the rate of photosynthesis is limited by the factor that is in shortest supply, relative to the demand.

$ \text{Rate of Photosynthesis} \propto \text{Minimum of [Light], [CO}_2\text{], Temperature, Water, etc.} $

Light

Light is essential for the light-dependent reactions.
Light Intensity:
- At low light intensities, the rate of photosynthesis increases linearly with increasing light intensity.
- At higher light intensities, the rate becomes limited by other factors. Light saturation usually occurs at 10% of full sunlight.
- Excessively high light intensity can damage the photosynthetic machinery (photo-oxidation), leading to a decrease in the rate of photosynthesis.
Light Quality (Wavelength): Photosynthesis occurs most effectively in the blue and red regions of the light spectrum (as shown by the action spectrum). Green light is least effective.
Duration of Light: Photosynthesis occurs only during the period of light exposure. Longer duration of light generally leads to more total photosynthesis, provided other factors are not limiting.

*(Image shows a graph with rate of photosynthesis on Y-axis and light intensity on X-axis, showing initial linear increase and then plateau)*

Carbon Dioxide Concentration

$CO_2$ is the major limiting factor for photosynthesis under normal conditions.
$CO_2$ Concentration:
- In the atmosphere, $CO_2$ concentration is around $0.03\%$ to $0.04\%$.
- The rate of photosynthesis increases with increasing $CO_2$ concentration up to a certain level.
- Above this optimal level, the rate may saturate or even decrease at very high concentrations.
- C3 plants respond to higher $CO_2$ concentrations up to about $0.05\%$, showing increased photosynthesis.
- C4 plants show saturation at much lower $CO_2$ concentrations (around $0.03\% - 0.04\%$). This is because their $CO_2$-concentrating mechanism is very efficient.

Graph comparing the effect of CO2 concentration on photosynthesis rate in C3 and C4 plants

*(Image shows a graph with rate of photosynthesis on Y-axis and CO2 concentration on X-axis, showing C4 plants saturating at lower CO2 and C3 plants saturating at higher CO2)*

Temperature

Temperature affects the enzyme-catalysed reactions of photosynthesis, particularly the dark reactions (Calvin cycle).
The rate of photosynthesis increases with temperature up to an optimum temperature.
Beyond the optimum temperature, enzyme activity decreases, and the rate of photosynthesis falls. High temperatures can denature enzymes.
Different plants have different optimum temperatures for photosynthesis. C4 plants generally have a higher optimum temperature than C3 plants, reflecting their adaptation to warmer climates.
Light reactions are less affected by temperature changes than dark reactions.

Water

Water is a reactant in photosynthesis (in the splitting of water during light reactions).
Water stress can significantly reduce the rate of photosynthesis by:
- Causing stomatal closure, which reduces $CO_2$ availability.
- Wilting of leaves, reducing the surface area for light absorption.
- Affecting metabolic activities directly.
While water is directly involved in the reaction, its effect as a limiting factor is usually indirect, primarily through its influence on stomatal opening.

Other internal factors affecting photosynthesis include the number, size, age, and orientation of leaves, mesophyll cells and chloroplasts, internal $CO_2$ concentration, and the amount of chlorophyll.