What Do We Know?

Previous experiments have shown that several factors are required for photosynthesis:

• **Chlorophyll:** The green pigment in leaves. Experiments with variegated leaves or leaves partially covered with black paper showed starch formation only in the green, exposed parts, indicating chlorophyll is essential.
• **Light:** Necessary for the process. Areas of leaves kept in the dark do not photosynthesise.
• **CO$_2$:** Required from the atmosphere. An experiment where part of a leaf is enclosed with a substance absorbing CO$_2$ (like KOH) shows that the part deprived of CO$_2$ does not form starch, while the exposed part does.

These simple experiments established that chlorophyll, light, and CO$_2$ are necessary for photosynthesis.

Early Experiments

Historical experiments played a crucial role in understanding photosynthesis:

• **Joseph Priestley (1770):** Showed the essential role of air. Using a bell jar, he observed that a burning candle or a mouse would damage the air (making it unfit for breathing/burning). When he placed a mint plant in the bell jar with the candle/mouse, they survived. He hypothesised that plants restore something to the air that animals and burning remove. He later discovered oxygen (1774).

• **Jan Ingenhousz (1730-1799):** Using a similar setup, showed that **sunlight is essential** for plants to purify air. With an aquatic plant in bright sunlight, he observed bubbles (later identified as oxygen) forming around the green parts, but not in the dark. This showed that only the green parts of plants release oxygen and that light is required.
• **Julius von Sachs (1854):** Provided evidence that **glucose is produced** during plant growth and is stored as starch. He showed that the green substance (chlorophyll) is located in chloroplasts, where glucose is made.
• **T.W Engelmann (1843–1909):** Used a prism to split light, illuminating a green alga (*Cladophora*) in a suspension of aerobic bacteria (used to detect O$_2$). Bacteria accumulated mainly in the regions of **blue and red light** of the spectrum. This described the first action spectrum of photosynthesis, roughly matching the absorption spectra of chlorophyll a and b.

By the mid-19th century, it was known that plants use light to make carbohydrates from CO$_2$ and water. The initial empirical equation was: CO$_2$ + H$_2$O $\xrightarrow{\text{Light}}$ [CH$_2$O] + O$_2$.

• **Cornelius van Niel (1897-1985):** Studied purple and green bacteria and demonstrated that photosynthesis is a light-dependent reaction where hydrogen from an oxidisable compound reduces CO$_2$ to carbohydrates (2H$_2$A + CO$_2$ $\xrightarrow{\text{Light}}$ 2A + [CH$_2$O] + H$_2$O). In green plants, H$_2$O is the hydrogen donor (H$_2$A), and it is oxidised to O$_2$ (A). In sulfur bacteria using H$_2$S, the oxidation product is sulfur (S) or sulfate, not O$_2$. He inferred that O$_2$ evolved by green plants comes from H$_2$O, not CO$_2$. This was later confirmed using radioisotopic techniques (using $^{18}$O).

The correct overall equation for photosynthesis is:

6CO$_2$ + 12H$_2$O $\xrightarrow{\text{Light}}$ C$_6$H$_{12}$O$_6$ + 6H$_2$O + 6O$_2$ (Glucose)

Photosynthesis is a complex multistep process, not a single reaction.

Where Does Photosynthesis Take Place?

Photosynthesis primarily occurs in the **green parts of plants**, mainly the **leaves**. Other green parts like stems can also photosynthesise. Within the plant cell, photosynthesis takes place in specialized organelles called **chloroplasts**.

Chloroplasts (studied in Chapter 8) have a membranous system consisting of **grana** (stacks of thylakoids) and **stroma lamellae** (connecting thylakoids), surrounded by a fluid matrix called the **stroma**. There is a clear division of labour within the chloroplast:

• The **membrane system (grana and stroma lamellae)** is the site of the **light reactions** (photochemical phase). This phase captures light energy and synthesises ATP and NADPH (high-energy chemical intermediates).
• The **stroma** is the site of the **dark reactions** (biosynthetic phase or carbon reactions). This phase uses the energy (ATP, NADPH) and reducing power produced during the light reactions to fix CO$_2$ and synthesise sugars (which are then converted to starch).

Dark reactions are not directly dependent on light but rely on the products of light reactions. They can continue for some time in the dark if ATP and NADPH are available.

How Many Types Of Pigments Are Involved In Photosynthesis?

The green colour of leaves is not due to a single pigment but a combination of several pigments. Chromatographic separation of leaf pigments reveals four main types:

• **Chlorophyll a:** Bright or blue-green. The **chief pigment** directly involved in the light reaction.
• **Chlorophyll b:** Yellow-green.
• **Xanthophylls:** Yellow.
• **Carotenoids:** Yellow to yellow-orange.

Pigments are substances that absorb light at specific wavelengths. The **absorption spectrum** of a pigment shows the wavelengths of light it absorbs. The **action spectrum** of photosynthesis shows the rate of photosynthesis at different wavelengths of light.

Comparing the absorption spectrum of chlorophyll a with the action spectrum of photosynthesis shows that maximum photosynthesis occurs in the blue and red regions of the spectrum, where chlorophyll a shows maximum absorption. This confirms chlorophyll a's primary role.

Chlorophyll b, xanthophylls, and carotenoids are **accessory pigments**. They absorb light at different wavelengths than chlorophyll a and transfer the absorbed energy to chlorophyll a. This allows a **wider range of wavelengths** of incoming light to be used for photosynthesis. Accessory pigments also protect chlorophyll a from **photo-oxidation** (damage by light).

What Is Light Reaction?

The **light reaction** (or photochemical phase) of photosynthesis is the process that captures light energy and converts it into chemical energy in the form of ATP and NADPH. It occurs in the thylakoid membranes of chloroplasts. Key events are light absorption, water splitting, oxygen release, and ATP and NADPH formation.

Pigments are organized into two discrete **photosystems (PS)**: **Photosystem I (PS I)** and **Photosystem II (PS II)**. These are named based on their discovery order.

Each photosystem has:

• A **light harvesting complex (LHC)** or antenna: Composed of hundreds of pigment molecules (chlorophyll b, xanthophylls, carotenoids, and most chlorophyll a) bound to proteins. They absorb light of different wavelengths and funnel the energy to the reaction center.
• A **reaction centre:** Consists of a single molecule of chlorophyll a. It is the site where the light energy is converted into chemical energy (electron excitation). The reaction center chlorophyll a is slightly different in the two photosystems based on its maximum absorption wavelength:
  • PS I reaction centre: Absorbs at 700 nm, called **P700**.
  • PS II reaction centre: Absorbs at 680 nm, called **P680**.

The Electron Transport

During the light reaction, excited electrons are transported through a series of electron carriers, leading to the formation of ATP and NADPH.

1. When PS II absorbs 680 nm red light, the electron in the P680 reaction center gets excited and is transferred to a primary electron acceptor.
2. The electron acceptor passes the electron to an **electron transport system (ETS)** consisting of cytochromes. This is an 'uphill' transfer to the acceptor, followed by a 'downhill' movement through the ETS in terms of redox potential.
3. The electrons from PS II are then transferred to the pigments of PS I.
4. Simultaneously, PS I absorbs 700 nm red light, exciting the electron in the P700 reaction center. This electron is transferred to another acceptor molecule with a higher redox potential.
5. These electrons from the PS I acceptor are then transferred 'downhill' to a molecule of NADP$^+$, reducing it to **NADPH + H$^+$** using the NADP reductase enzyme.

This entire process of electron transfer, from water splitting to PS II, through the ETS to PS I, and finally to NADP$^+$ reduction, follows a zig-zag path when carriers are placed on a redox potential scale. This is called the **Z scheme** of electron transport.

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Splitting of Water (Photolysis)

To continuously supply electrons to PS II, water molecules are split. The **splitting of water (photolysis)** is associated with **PS II** and occurs on the **inner side of the thylakoid membrane**. Water is split into protons (H$^+$), oxygen ([O], which combines to form O$_2$), and electrons. The electrons produced replace the electrons lost by PS II.

2H$_2$O $\to$ 4H$^+$ + O$_2$ + 4e$^-$

The oxygen released from water splitting is one of the net products of photosynthesis and diffuses out of the chloroplast.

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Cyclic and Non-cyclic Photo-phosphorylation

**Photophosphorylation** is the synthesis of ATP from ADP and inorganic phosphate (Pi) in the presence of light. This process occurs in chloroplasts (and mitochondria, which is oxidative phosphorylation).

• **Non-cyclic photophosphorylation:** Occurs when **both PS II and PS I** work in a series, coupled by the electron transport chain (Z scheme). Electrons flow linearly from water splitting through PS II, ETS, PS I, and finally to NADP$^+$. This process synthesises **both ATP and NADPH + H$^+$**.
• **Cyclic photophosphorylation:** Occurs when **only PS I** is functional. Electrons from PS I are excited but are cycled back to the PS I complex through the electron transport chain (instead of going to NADP$^+$). This process synthesises **only ATP**, not NADPH + H$^+$. It is thought to occur in the stroma lamellae, which contain PS I but lack PS II and NADP reductase enzyme. Cyclic photophosphorylation also occurs when light wavelengths beyond 680 nm (which PS II cannot absorb efficiently) are available.

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Chemiosmotic Hypothesis

The **chemiosmotic hypothesis** explains how ATP is synthesized in chloroplasts (and mitochondria). ATP synthesis is linked to the development of a **proton gradient** across a membrane. In chloroplasts, this gradient forms across the **thylakoid membrane**, with protons accumulating in the **lumen** (inside the thylakoid) and decreasing in the stroma (outside the thylakoid membrane).

Factors causing the proton gradient across the thylakoid membrane:

1. **Splitting of water:** Water splitting on the **inner side** of the thylakoid membrane releases protons directly into the lumen.
2. **Electron transport:** As electrons move through the electron transport chain between PS II and PS I, protons are transported across the membrane. A molecule transferring electrons to a carrier on the inner side takes up a proton from the stroma and releases it into the lumen.
3. **NADP$^+$ reduction:** The NADP reductase enzyme on the **stroma side** of the membrane uses electrons and protons from the stroma to reduce NADP$^+$ to NADPH + H$^+$. This removes protons from the stroma, further contributing to the gradient.

The accumulated protons in the lumen create a proton gradient and a lower pH inside the thylakoid compared to the stroma. This gradient is a source of energy. ATP is synthesised as protons move down their concentration gradient from the lumen to the stroma through a transmembrane channel formed by the **CF$_0$ part of the ATP synthase enzyme**. The **CF$_1$ part** of ATP synthase, which protrudes into the stroma, uses the energy from proton flow to catalyse the synthesis of ATP from ADP and Pi.

Chemiosmosis requires a membrane, a proton pump (electron transport chain), a proton gradient, and ATP synthase. The ATP and NADPH produced in the light reaction are used immediately in the biosynthetic phase (dark reaction) in the stroma to fix CO$_2$ and synthesise sugars.

Where Are The ATP And NADPH Used?

The ATP and NADPH produced during the light reaction are used in the **biosynthetic phase** (dark reaction) of photosynthesis. This phase occurs in the **stroma** of the chloroplasts and involves the fixation of CO$_2$ and synthesis of sugars. It does not directly require light but depends on the ATP and NADPH from the light reaction. If the supply of ATP and NADPH stops (e.g., if light is removed), the biosynthetic phase also stops after some time.

The biosynthetic phase uses CO$_2$ and H$_2$O to produce sugars ([CH$_2$O]n). Scientists studied how CO$_2$ is fixed and identified the pathway called the **Calvin cycle** (or C$_3$ pathway) using radioactive $^{14}$C by Melvin Calvin. The first stable product of CO$_2$ fixation identified was a 3-carbon organic acid, **3-phosphoglyceric acid (PGA)**.

Later, in some other plants, the first stable product of CO$_2$ fixation was found to be a 4-carbon organic acid, **oxaloacetic acid (OAA)**. These plants utilise the **C$_4$ pathway**. This led to the classification of plants based on the first product of CO$_2$ fixation: C$_3$ plants (first product is a C$_3$ acid, PGA) and C$_4$ plants (first product is a C$_4$ acid, OAA).

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The Primary Acceptor of CO$_2$

Scientists worked to identify the molecule that initially accepts CO$_2$ in the Calvin cycle. Surprisingly, the CO$_2$ acceptor molecule was found to be a 5-carbon ketose sugar, **ribulose-1,5-bisphosphate (RuBP)**, not a 2-carbon compound as initially thought.

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The Calvin Cycle (C$_3$ Pathway)

The Calvin cycle is the main biosynthetic pathway in **all photosynthetic plants** (both C$_3$ and C$_4$) for synthesising sugars from CO$_2$. It is a cyclic process where RuBP is regenerated. It occurs in the stroma.

The Calvin cycle proceeds in three main stages:

1. **Carboxylation:** The crucial step where CO$_2$ is fixed. CO$_2$ combines with the 5-carbon RuBP molecule. This reaction is catalysed by the enzyme **RuBP carboxylase-oxygenase (RuBisCO)**. This results in the formation of two molecules of the 3-carbon PGA (the first stable product of the Calvin cycle).

2. **Reduction:** A series of reactions that convert PGA into glucose (sugars). This stage **utilises ATP and NADPH** produced during the light reaction. For every CO$_2$ molecule fixed, 2 molecules of ATP are used for phosphorylation, and 2 molecules of NADPH are used for reduction in this stage.
3. **Regeneration:** The steps that regenerate the CO$_2$ acceptor molecule, RuBP, from triose phosphate. This regeneration is essential for the cycle to continue and **requires 1 molecule of ATP** per CO$_2$ molecule fixed.

For every CO$_2$ molecule entering the Calvin cycle, a total of **3 molecules of ATP** (2 in reduction, 1 in regeneration) and **2 molecules of NADPH** (in reduction) are required. To form one molecule of glucose (a 6-carbon sugar, requiring the fixation of 6 CO$_2$ molecules), the Calvin cycle must turn 6 times. Therefore, to make one glucose molecule, the requirements are $6 \times 3 = 18$ ATP molecules and $6 \times 2 = 12$ NADPH molecules.

The C$_4$ Pathway (Hatch and Slack Pathway)

Plants adapted to dry tropical regions (C$_4$ plants) have evolved a mechanism to increase CO$_2$ concentration at the site of the Calvin cycle, minimizing photorespiration. They use the C$_4$ pathway as an initial step for CO$_2$ fixation, followed by the Calvin cycle for sugar synthesis.

C$_4$ plants have special characteristics:

• **Special leaf anatomy (Kranz anatomy):** Characterised by large bundle sheath cells around the vascular bundles, forming several layers. These cells are prominent, have many chloroplasts, thick walls impervious to gas exchange, and no intercellular spaces. Mesophyll cells are located outside the bundle sheath cells.
• Higher tolerance to temperatures and light intensities.
• Lack photorespiration (or have negligible photorespiration).
• Greater productivity of biomass compared to C$_3$ plants.

The C$_4$ pathway, also called the **Hatch and Slack Pathway**, operates in two types of cells in C$_4$ leaves: **mesophyll cells** and **bundle sheath cells**.

1. **Initial Carboxylation (in Mesophyll cells):** The primary CO$_2$ acceptor is a 3-carbon molecule, **phosphoenol pyruvate (PEP)**. CO$_2$ is fixed by the enzyme **PEP carboxylase (PEPcase)**, which is present in mesophyll cells but lacks RuBisCO. The first stable product is the 4-carbon acid, **oxaloacetic acid (OAA)**.
2. **Transport of C$_4$ acid (from Mesophyll to Bundle Sheath cells):** OAA is converted into other C$_4$ acids (malic acid or aspartic acid) in the mesophyll cells. These are transported to the bundle sheath cells.
3. **Decarboxylation (in Bundle Sheath cells):** In the bundle sheath cells, the C$_4$ acids are broken down (decarboxylated) to release **CO$_2$** and a 3-carbon molecule. The bundle sheath cells contain **RuBisCO** but lack PEPcase.
4. **Calvin Cycle (in Bundle Sheath cells):** The high concentration of CO$_2$ released in the bundle sheath cells enters the Calvin cycle (C$_3$ pathway), which is common to all plants, for sugar synthesis. The high CO$_2$ concentration ensures that RuBisCO acts primarily as a carboxylase, minimizing photorespiration.
5. **Regeneration of PEP (from Bundle Sheath to Mesophyll cells):** The 3-carbon molecule produced in the bundle sheath cells is transported back to the mesophyll cells, where it is converted back to PEP, completing the C$_4$ cycle.

Thus, the C$_4$ pathway acts as a mechanism to concentrate CO$_2$ in the bundle sheath cells where the Calvin cycle takes place. This spatial separation of initial CO$_2$ fixation and the Calvin cycle is a key feature of C$_4$ plants.

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hotorespiration

**Photorespiration** is a wasteful process that occurs in C$_3$ plants. It involves the enzyme RuBisCO acting as an oxygenase under certain conditions. When the concentration of O$_2$ is relatively high compared to CO$_2$ in the C$_3$ chloroplast stroma, RuBisCO binds to O$_2$ instead of CO$_2$.

Reaction of RuBisCO with O$_2$:

RuBP + O$_2$ $\xrightarrow{\text{RuBisCO}}$ Phosphoglycerate (3C) + Phosphoglycolate (2C)

In the photorespiratory pathway that follows, the 2-carbon phosphoglycolate is metabolized, releasing CO$_2$ in the process. Photorespiration occurs in chloroplasts, peroxisomes, and mitochondria. It **does not produce sugars, ATP, or NADPH**. Instead, it **consumes ATP** and results in the **release of CO$_2$** that was previously fixed.

The biological function of photorespiration is not fully understood, but it is considered a wasteful process that reduces the efficiency of photosynthesis in C$_3$ plants, especially under conditions of high light intensity, high temperature, and low CO$_2$ concentration (conditions that favour RuBisCO oxygenase activity).

C$_4$ plants effectively suppress photorespiration by concentrating CO$_2$ in the bundle sheath cells, ensuring RuBisCO acts as a carboxylase.

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Factors affecting Photosynthesis

The rate of photosynthesis is influenced by several factors, categorized as internal (plant factors) and external (environmental factors).

• **Internal factors:** Number, size, age, and orientation of leaves, mesophyll cells and chloroplasts within leaves, internal CO$_2$ concentration, and amount of chlorophyll. These are influenced by the plant's genetics and growth.
• **External factors:** Availability of sunlight, temperature, CO$_2$ concentration, and water.

Photosynthesis is a process affected by multiple factors simultaneously. According to **Blackman's Law of Limiting Factors (1905)**, when a process is affected by more than one factor, its rate is determined by the factor that is nearest to its minimum value. This limiting factor is the one whose change directly affects the rate.

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Light

Light affects photosynthesis in terms of its quality (wavelength), intensity, and duration. At low light intensities, there's a linear relationship between incident light and CO$_2$ fixation rate – increasing light increases the rate. At higher light intensities, the rate increases more slowly and eventually plateaus as other factors become limiting (light saturation).

Light saturation usually occurs at about 10% of full sunlight. Thus, light is rarely a limiting factor in nature except for plants in shade or dense forests. Very high light intensities can damage chlorophyll (photo-oxidation), decreasing the photosynthesis rate.

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Carbon dioxide Concentration

CO$_2$ is the most significant limiting factor for photosynthesis in most plants under normal conditions, as its atmospheric concentration (0.03-0.04%) is low. Increasing CO$_2$ concentration up to about 0.05% can increase CO$_2$ fixation rates. Levels beyond that can be damaging over long periods.

C$_3$ and C$_4$ plants respond differently to CO$_2$ concentration. At high light intensities, C$_4$ plants show CO$_2$ saturation at lower concentrations ($\approx$ 360 μLL$^{-1}$) than C$_3$ plants ($\approx$ 450 μLL$^{-1}$ or more). C$_3$ plants show increased photosynthesis rates with increasing CO$_2$ concentration up to this higher saturation point, indicating that current atmospheric CO$_2$ levels limit photosynthesis in C$_3$ plants. This is why CO$_2$ enrichment in greenhouses can increase yields for C$_3$ crops.

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Temperature

Photosynthesis involves enzymatic reactions (especially in the dark phase), which are temperature-sensitive. Light reactions are also affected but to a lesser extent. The rate of photosynthesis has an optimum temperature.

Temperature optimum varies between plants: C$_4$ plants and tropical plants have a higher temperature optimum for photosynthesis than C$_3$ plants and plants from temperate climates. Activity is lower below or above the optimum.

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Water

Water is a reactant in photosynthesis (light reaction), but its effect on the rate is often indirect, mainly through its influence on the plant's overall health and stomatal opening. Water stress (scarcity of water) causes stomata to close to conserve water, which reduces the uptake of CO$_2$, thus limiting photosynthesis. Water stress also causes wilting of leaves, reducing the surface area available for light absorption and decreasing metabolic activity.

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