Chapter Notes

Photosynthesis in Higher Plants

Photosynthesis is the fundamental process by which green plants create their own food. They are called autotrophs because they synthesize the food they need. All other organisms, including humans, that depend on plants for food are called heterotrophs.

Photosynthesis is a physico-chemical process where green plants use light energy to synthesize organic compounds (food). It is the basis of all life on Earth for two main reasons:

1. It is the primary source of all food on the planet.
2. It releases oxygen into the atmosphere, which is essential for respiration.

What do we Know?

Simple experiments demonstrate the basic requirements for photosynthesis:

• Chlorophyll and Light: An experiment with a variegated (partly green, partly white) leaf shows that starch (a product of photosynthesis) is formed only in the green parts of the leaf and only when exposed to light.
• Carbon Dioxide ($CO_2$): If a part of a leaf is enclosed in a test tube with potassium hydroxide (KOH), which absorbs $CO_2$, that part of the leaf will not form starch, even in the presence of light. This proves that $CO_2$ is necessary for photosynthesis.

Early Experiments

Our understanding of photosynthesis developed through a series of key experiments over centuries.

• Joseph Priestley (1770): He conducted experiments using a bell jar, a candle, and a mouse. He observed that a burning candle would soon go out, and a mouse would suffocate, in a sealed bell jar. He concluded that both burning and breathing "damage the air." However, when he placed a mint plant in the bell jar with the candle or the mouse, the candle continued to burn and the mouse stayed alive.

  • Hypothesis: Plants restore to the air whatever breathing animals and burning candles remove. Priestley is credited with discovering oxygen in 1774.

• Jan Ingenhousz (1779): Using a similar setup, he showed that sunlight is essential for this process. He experimented with an aquatic plant and noticed that in bright sunlight, small bubbles formed around the green parts, but not in the dark. He later identified these bubbles as oxygen.

  • Conclusion: Only the green parts of plants can release oxygen, and they do so only in the presence of sunlight.

• Julius von Sachs (1854): He provided evidence that plants produce glucose during growth, which is usually stored as starch. He also discovered that the green substance, now known as chlorophyll, is located in special bodies within plant cells, later called chloroplasts.

  • Conclusion: Glucose is made in the green parts of the plant and stored as starch.

• T.W Engelmann (1843-1909): He performed an ingenious experiment by splitting light into its spectral components (a rainbow) using a prism and shining it on a green alga, Cladophora. He used aerobic bacteria, which are attracted to oxygen, to detect where photosynthesis was happening most.

  • Observation: The bacteria accumulated mainly in the blue and red light regions. This was the first action spectrum of photosynthesis, showing which wavelengths of light are most effective for the process.

• Cornelius van Niel (1897-1985): A microbiologist who studied purple and green sulfur bacteria. He demonstrated that photosynthesis is a light-dependent reaction where hydrogen from a suitable compound reduces carbon dioxide to carbohydrates.

  • His general equation was: $2H_2A + CO_2 \xrightarrow{\text{Light}} 2A + CH_2O + H_2O$
  • In green plants, the hydrogen donor is water ($H_2O$), which is oxidized to oxygen ($O_2$).
  • In purple and green sulfur bacteria, the hydrogen donor is hydrogen sulfide ($H_2S$), and the product is sulfur, not oxygen.
  • Crucial Inference: The oxygen evolved by green plants comes from the splitting of water ($H_2O$), not from carbon dioxide ($CO_2$). This was later confirmed using radioisotope techniques.

The correct, balanced chemical equation for photosynthesis in higher plants is: $6CO_2 + 12H_2O \xrightarrow{\text{Light}} C_6H_{12}O_6 + 6H_2O + 6O_2$ Here, $C_6H_{12}O_6$ represents glucose.

Where does Photosynthesis take Place?

Photosynthesis occurs in the green parts of plants, primarily in the leaves, within specialized cells called mesophyll cells. These cells contain a large number of chloroplasts, which are the actual sites of photosynthesis.

Inside a chloroplast, there is a clear division of labor:

• Membranous System (Grana and Stroma Lamellae): This system is responsible for trapping light energy and synthesizing energy-rich molecules like ATP and NADPH. These reactions are directly driven by light and are called the light reactions or photochemical reactions.
• Matrix Stroma: This is the fluid-filled space within the chloroplast. It contains the enzymes required to synthesize sugars (food). These reactions are not directly light-driven but depend on the products of the light reactions (ATP and NADPH). They are called the dark reactions or carbon reactions.

How many Types of Pigments are Involved in Photosynthesis?

The green color of leaves is not due to a single pigment but a combination of four, which can be separated using paper chromatography:

• Chlorophyll a (bright or blue-green)
• Chlorophyll b (yellow-green)
• Xanthophylls (yellow)
• Carotenoids (yellow to yellow-orange)

Pigments are substances that can absorb light at specific wavelengths.

• Chlorophyll a is the most abundant and is considered the chief pigment because it is directly involved in converting light energy to chemical energy. The absorption spectrum of chlorophyll a shows that it absorbs light most strongly in the blue and red regions of the visible spectrum. The rate of photosynthesis is also highest in these regions.
• Chlorophyll b, xanthophylls, and carotenoids are called accessory pigments. Their roles are to:
  1. Absorb light at different wavelengths and transfer the energy to chlorophyll a, thus broadening the range of light that can be used for photosynthesis.
  2. Protect chlorophyll a from photo-oxidation (damage by excessive light).

What is Light Reaction?

The light reactions, or the 'Photochemical' phase, include:

• Light absorption
• Water splitting (photolysis)
• Oxygen release
• Formation of high-energy chemical intermediates: ATP and NADPH.

The pigments are organized into two distinct complexes called Photosystem I (PS I) and Photosystem II (PS II).

• Each photosystem contains hundreds of pigment molecules that form a light-harvesting complex (LHC), also called antennae. These molecules absorb light energy and funnel it to a special, single molecule of chlorophyll a, which forms the reaction centre.
• The reaction centre of PS I is called P700 because its chlorophyll a has an absorption peak at 700 nm.
• The reaction centre of PS II is called P680 because its chlorophyll a has an absorption peak at 680 nm.

The Electron Transport

The transport of electrons during the light reaction follows a specific pathway known as the Z scheme.

1. Excitation of PS II: The P680 reaction centre of PS II absorbs red light (680 nm), causing an electron to become excited and jump to a higher energy level.
2. Electron Acceptor: This high-energy electron is captured by an electron acceptor.
3. Electron Transport System (ETS): The electron is then passed down an electron transport system consisting of cytochromes. This movement is "downhill" in terms of redox potential, releasing energy.
4. Transfer to PS I: The electron is passed on to the PS I reaction centre.
5. Excitation of PS I: Simultaneously, the P700 reaction centre of PS I absorbs red light (700 nm), exciting another electron.
6. Transfer to NADP+: This electron is transferred to another acceptor and finally to the energy-rich molecule NADP+, reducing it to NADPH + H+.

Splitting of Water

To replace the electron lost from PS II, water is split. This process is associated with PS II and occurs on the inner side of the thylakoid membrane. $2H_2O \longrightarrow 4H^+ + O_2 + 4e^-$

• The electrons ($e^-$) are used to replace those lost by P680.
• The protons ($H^+$) accumulate in the thylakoid lumen.
• Oxygen ($O_2$) is released as a byproduct of photosynthesis.

Cyclic and Non-cyclic Photo-phosphorylation

Phosphorylation is the process of synthesizing ATP. When this is driven by light energy, it is called photophosphorylation.

• Non-cyclic Photo-phosphorylation:

  • This is the Z-scheme described above.
  • It involves both PS II and PS I working in series.
  • It produces both ATP and NADPH + H+.

• Cyclic Photo-phosphorylation:

  • Occurs when only PS I is functional, possibly in the stroma lamellae which lack PS II and the NADP reductase enzyme.
  • The excited electron from P700 is cycled back to the PS I complex through the electron transport chain, instead of being passed to NADP+.
  • This process produces only ATP, not NADPH.
  • It also occurs when only light of wavelengths beyond 680 nm is available.

Chemiosmotic Hypothesis

This hypothesis explains how ATP is synthesized in the chloroplast. It relies on the creation of a proton gradient across the thylakoid membrane.

A high concentration of protons ($H^+$) accumulates in the thylakoid lumen, while the concentration in the stroma decreases. This gradient is created by three processes:

1. Splitting of Water: Water is split on the inner side of the membrane, releasing protons into the lumen.
2. Proton Pumping: As electrons move through the ETS, a primary electron acceptor (located on the outer side) transfers its electron to an H carrier. This carrier picks up a proton from the stroma, transports it across the membrane, and releases it into the lumen.
3. NADP+ Reduction: The NADP reductase enzyme is on the stroma side. It removes protons from the stroma to reduce NADP+ to NADPH + H+.

This proton gradient is a form of stored energy. The gradient is broken down when protons move from the lumen back to the stroma through a transmembrane channel in an enzyme called ATP synthase.

The ATP synthase enzyme has two parts:

• $CF_0$: Embedded in the thylakoid membrane, forming a channel for protons.
• $CF_1$: Protrudes on the stroma side. The energy released by the flow of protons causes a conformational change in $CF_1$, which drives the synthesis of ATP from ADP and inorganic phosphate.

Where are the ATP and NADPH Used?

The products of the light reactions (ATP, NADPH) are used in the biosynthetic phase (dark reactions) to synthesize sugars by fixing atmospheric $CO_2$. This process occurs in the stroma.

Melvin Calvin used radioactive carbon-14 ($^{14}C$) to trace the path of carbon in algal photosynthesis. He discovered that the first product of $CO_2$ fixation was a 3-carbon acid, 3-phosphoglyceric acid (PGA). This pathway is called the Calvin cycle.

• Plants where the first product is a 3-carbon acid (PGA) are called $C_3$ plants.
• In some other plants, the first stable product was found to be a 4-carbon acid, oxaloacetic acid (OAA). These are called $C_4$ plants.

The Primary Acceptor of $CO_2$

In the Calvin cycle, the molecule that accepts the incoming $CO_2$ is a 5-carbon ketose sugar called ribulose bisphosphate (RuBP).

The Calvin Cycle

The Calvin cycle occurs in all photosynthetic plants, whether they are $C_3$ or $C_4$. It has three main stages:

1. Carboxylation:

   • This is the fixation of $CO_2$.
   • $CO_2$ combines with RuBP to form an unstable 6-carbon intermediate, which immediately splits into two molecules of 3-PGA.
   • This crucial step is catalyzed by the enzyme RuBP carboxylase-oxygenase, or RuBisCO.

2. Reduction:

   • A series of reactions where the 3-PGA is converted into carbohydrate (sugar).
   • This process uses the ATP and NADPH produced during the light reactions.
   • For every one molecule of $CO_2$ fixed, 2 molecules of ATP and 2 molecules of NADPH are used.

3. Regeneration:

   • The $CO_2$ acceptor, RuBP, is regenerated to keep the cycle going.
   • This step requires one molecule of ATP for phosphorylation.

• In: 6 $CO_2$ + 18 ATP + 12 NADPH
• Out: 1 Glucose + 18 ADP + 12 NADP

The $C_4$ Pathway

Plants adapted to dry, tropical regions (like maize and sorghum) have the $C_4$ pathway. These plants are special because they:

• Have a special leaf anatomy called 'Kranz' anatomy.
• Tolerate higher temperatures and light intensities.
• Lack photorespiration, leading to greater productivity.

Kranz Anatomy: The vascular bundles in the leaves are surrounded by large cells called bundle sheath cells, which form a "wreath" ('Kranz' in German). These cells have thick walls and a large number of chloroplasts.

The $C_4$ pathway, also known as the Hatch and Slack Pathway, is a mechanism to concentrate $CO_2$ for the Calvin cycle.

1. Initial $CO_2$ Fixation (in Mesophyll Cells):

   • The primary $CO_2$ acceptor is a 3-carbon molecule, phosphoenolpyruvate (PEP).
   • The enzyme is PEP carboxylase (PEPcase), which has a high affinity for $CO_2$. Mesophyll cells in $C_4$ plants lack RuBisCO.
   • The product is the 4-carbon acid, oxaloacetic acid (OAA).

2. Transport:

   • OAA is converted to other 4-carbon acids like malic acid or aspartic acid.
   • These acids are transported from the mesophyll cells to the bundle sheath cells.

3. Decarboxylation (in Bundle Sheath Cells):

   • Inside the bundle sheath cells, the 4-carbon acid is broken down, releasing $CO_2$ and a 3-carbon molecule.
   • This release of $CO_2$ raises its concentration significantly around the RuBisCO enzyme.
   • The released $CO_2$ then enters the Calvin cycle (the C3 pathway) to produce sugars.

4. Regeneration:

   • The 3-carbon molecule is transported back to the mesophyll cells, where it is converted back into PEP, using ATP.

Photorespiration

RuBisCO, the most abundant enzyme in the world, has a dual function. Its active site can bind to both $CO_2$ (carboxylation) and $O_2$ (oxygenation). This binding is competitive.

• In $C_3$ plants, when $O_2$ concentration is high, RuBisCO binds to $O_2$ instead of $CO_2$.

• Instead of forming two molecules of 3-PGA, the RuBP combines with $O_2$ to form one molecule of phosphoglycerate (3-C) and one molecule of phosphoglycolate (2-C).

• This pathway is called photorespiration. It is considered a wasteful process because:

  • It does not produce any sugar.
  • It does not produce ATP or NADPH.
  • It actually consumes ATP and results in the release of $CO_2$.

• In $C_4$ plants, photorespiration does not occur. This is because the $C_4$ pathway acts as a $CO_2$-concentrating mechanism. It pumps $CO_2$ into the bundle sheath cells, ensuring that the concentration of $CO_2$ at the RuBisCO enzyme site is high, which minimizes the oxygenase activity. This lack of photorespiration is a major reason why $C_4$ plants have higher productivity and yields, especially in hot, dry climates.

Factors affecting Photosynthesis

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

Blackman's (1905) Law of Limiting Factors: If a chemical process is affected by more than one factor, its rate will be determined by the factor that is nearest to its minimal value. This factor is called the limiting factor.

Light

• At low light intensities, the rate of photosynthesis is directly proportional to the light intensity.
• At higher light intensities, the rate does not increase further because other factors (like $CO_2$ concentration) become limiting. This is called light saturation.
• Light saturation typically occurs at about 10% of full sunlight, so light is rarely a limiting factor in nature, except for plants in shade or dense forests.
• Excessively high light can cause the breakdown of chlorophyll, decreasing the rate of photosynthesis.

Carbon dioxide Concentration

• $CO_2$ is the major limiting factor for photosynthesis, as its concentration in the atmosphere is very low (0.03% to 0.04%).
• An increase in $CO_2$ concentration (up to 0.05%) can increase the rate of photosynthesis.
• $C_3$ and $C_4$ plants respond differently:
  • $C_4$ plants show saturation at about 360 $\mu L L^{-1}$.
  • $C_3$ plants respond to higher $CO_2$ concentrations and show saturation only beyond 450 $\mu L L^{-1}$. This means that for $C_3$ plants, the current atmospheric $CO_2$ level is limiting.
• This property is used in greenhouses to grow crops like tomatoes and bell peppers in a $CO_2$-enriched atmosphere to increase yield.

Temperature

• The dark reactions are enzymatic and therefore temperature-controlled.
• $C_3$ plants have a lower temperature optimum (around 20-25°C).
• $C_4$ plants respond to higher temperatures and have a higher temperature optimum (around 30-40°C).
• The optimal temperature also depends on the plant's habitat.

Water

• Water is a reactant in the light reaction, but its effect on photosynthesis is more indirect.
• Water stress (lack of water) causes the stomata to close to conserve water. This reduces the availability of $CO_2$ for photosynthesis.
• Water stress also causes leaves to wilt, reducing their surface area and metabolic activity.