Photosynthesis in Higher Plants Biology Notes - Experiments, Light Reaction, Calvin Cycle, C3 and C4 Pathway and Factors Affecting Photosynthesis

• Photosynthesis is a physico-chemical process by which green plants use light energy (solar energy) to synthesize organic compounds, making them autotrophs.

• It is the basis of life on earth, as all living forms ultimately depend on sunlight for energy.

Importance of Photosynthesis

• It is the primary source of all food on earth.
• It releases oxygen into the atmosphere.

Experiments Related to Photosynthesis

1. Variegated Leaf Experiment

• Take a variegated leaf (or a leaf partially covered with black paper) exposed to light.

• Test the leaf for starch. The results show that photosynthesis occurs only in the green parts of the leaves in the presence of light.

2. Half-Leaf Experiment

• Enclose part of a leaf in a test tube containing KOH-soaked cotton (which absorbs CO₂).

• Expose the other half of the leaf to air and place the setup in light for some time.

• Test the leaf for starch. The exposed part tests positive for starch, while the portion in the tube tests negative, proving that CO₂ is required for photosynthesis.

Early Experiments

Experiments by Joseph Priestley (1770)

• Priestley performed experiments to prove the role of air in the growth of green plants and discovered oxygen in 1774.

• He observed that a candle burning in a closed bell jar gets extinguished, and a mouse suffocates in a closed jar, concluding that a burning candle or breathing animal damages the air.

• By placing a mint plant in the same bell jar, he found that the mouse stayed alive and the candle continued to burn. He hypothesized that plants restore air damaged by animals or candles.

  

Experiments by Jan Ingenhousz (1730–1799)

• Ingenhousz conducted Priestley’s experiment in darkness and sunlight, showing that sunlight is essential for plants to purify air fouled by candles or animals.

• Using an aquatic plant, he observed that in bright sunlight, small bubbles formed around green parts, but not in the dark. He identified these bubbles as oxygen, proving that only green parts of plants release O₂.

Experiments by Julius von Sachs (1854)

Sachs proved that:

• Glucose is produced during plant growth and usually stored as starch.
• Chlorophyll is located in chloroplasts.
• Glucose is made in the green parts of plants.

Experiments by T.W. Engelmann (1843–1909)

• Engelmann split light using a prism and illuminated a green alga Cladophora) in a suspension of aerobic bacteria to detect O₂ evolution sites.

• Bacteria accumulated mainly in the blue and red light regions of the spectrum, describing the first action spectrum of photosynthesis, resembling the absorption spectra of chlorophyll a and b.

• By the mid-19th century, it was known that plants use light energy to make carbohydrates from CO₂ and H₂O. The empirical equation is:

Where [CH₂O] represents a carbohydrate.

Experiments by Cornelius van Niel

• Van Niel studied purple and green bacteria, demonstrating that photosynthesis is a light-dependent reaction where hydrogen from an oxidizable compound reduces CO₂ to carbohydrates.

• In plants, H₂O is the hydrogen donor, oxidized to O₂. In purple and green sulfur bacteria, H₂S is the hydrogen donor, producing sulfur or sulfate, not O₂.

• Van Niel inferred that O₂ evolved by green plants comes from H₂O, not CO₂, later confirmed by radioisotopic techniques. The correct equation for photosynthesis is:

Photosynthesis: Site & Pigments

• Photosynthesis occurs in green leaves and other green parts, within chloroplasts in the walls of mesophyll cells of leaves, optimizing the capture of incident light.

• Chloroplasts contain a membranous system consisting of grana, stroma lamellae, and matrix stroma.

• Each granum is a group of membrane-bound sacs called thylakoids (lamellae), which contain leaf pigments.

• The membrane system traps light energy to synthesize ATP and NADPH during light reactions.

• In the stroma, enzymatic reactions synthesize sugar, forming starch during dark reactions (carbon reactions), which are light-dependent despite the name.

Pigments Involved in Photosynthesis

• Pigments absorb light at specific wavelengths. Chromatography reveals the following leaf pigments:

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

• Chlorophyll b, xanthophylls, and carotenoids are accessory pigments.

• Functions of accessory pigments:

• Absorb light at different wavelengths and transfer energy to chlorophyll a.
• Protect chlorophyll a from photo-oxidation.

• The absorption spectrum and action spectrum coincide closely, showing maximum photosynthesis in the blue and red regions of the spectrum, with chlorophyll a as the chief pigment.

Photosystems

• Pigments are organized into two photosystems: Photosystem I (PSI) and Photosystem II (PSII), named in order of discovery.

• Each photosystem includes chlorophyll a and accessory pigments bound by proteins.

• All pigments except one chlorophyll a molecule form a light-harvesting complex (LHC or antennae).

• A single chlorophyll a acts as the reaction center.

• In PS I, the reaction center absorbs light at 700 nm, called P700.

• In PS II, the reaction center absorbs light at 680 nm, called P680.

Light Reaction (Photochemical Phase)

Light reactions involve light absorption, water splitting, oxygen release, and the formation of ATP and NADPH (high-energy chemical intermediates).

The Electron Transport

• When PS II absorbs red light of 680 nm, electrons are excited and transferred to an electron acceptor.

• The electron acceptor passes them to a chain of the electron transport system consisting of cytochromes.

• This movement is downhill in terms of redox potential scale.

• The electrons are then transferred to the pigments of PS I.

• Simultaneously, electrons in PS I are excited by red light of 700 nm and transferred to another acceptor molecule with a greater redox potential.

• These electrons move downhill to NADP⁺, reducing it to NADPH + H⁺.

• The transfer of electrons from PS II to PS I and finally to NADP⁺ is called the Z scheme, named for its zigzag shape on a redox potential scale.

Splitting of Water (Photolysis)

• The water-splitting complex in PS II is located on the inner side of the thylakoid membrane.

2H₂O → 4H⁺ + O₂ + 4e⁻

• PS II continuously supplies electrons by replacing those from water splitting, providing electrons to replace those removed from PS I.

• The protons (H⁺) are used to reduce NADP⁺ to NADPH.

• Oxygen is released as a by-product of photosynthesis.

Photophosphorylation

• Phosphorylation is the synthesis of ATP by cells in mitochondria and chloroplasts.

• Photophosphorylation is ATP synthesis from ADP in chloroplasts in the presence of light, occurring in two ways: non-cyclic and cyclic.

a) Non-Cyclic Photophosphorylation

• Occurs when PS II and PS I work in series through an electron transport chain, as in the Z scheme.

• Both ATP and NADPH + H⁺ are synthesized.

• It is non-cyclic because electrons lost by PS II do not return to it but pass to NADP⁺.

b) Cyclic Photophosphorylation

• Occurs in stroma lamellae when only PS I is functional.

• The electron circulates within the photosystem, and ATP synthesis occurs due to the cyclic flow of electrons.

• Grana lamellae have both PS I and PS II, but stroma lamellae lack PS II and NADP reductase.

• Electrons do not pass to NADP⁺ but cycle back to PS I through the electron transport chain.

• Only ATP is synthesized (no NADPH + H⁺).

• Cyclic photophosphorylation occurs when only light wavelengths beyond 680 nm are available.

Chemiosmotic Hypothesis

• The chemiosmotic hypothesis explains the mechanism of ATP synthesis in chloroplasts.

• Chemiosmosis is the movement of ions across a semipermeable membrane, occurring in chloroplasts and mitochondria.

• It requires a membrane, a proton pump, a proton gradient across thylakoid membranes, and ATP synthase.

• Water splitting on the inner side of the membrane causes protons to accumulate in the thylakoid lumen.

• Protons are transported across the membrane as electrons move through photosystems, due to:

• The primary electron acceptor, located on the outer side of the membrane, transfers electrons to an H carrier, removing a proton from the stroma. When the electron is passed to an electron carrier on the inner side, a proton is released into the lumen.
• The NADP reductase enzyme, on the stroma side, requires protons from the stroma along with electrons from PS I to reduce NADP⁺.

• This decreases protons in the stroma and accumulates them in the lumen, creating a proton gradient and lowering the pH in the lumen.

• The breakdown of the proton gradient drives ATP synthesis via the ATP synthase enzyme, which consists of:

• CF₀: Embedded in the membrane, forming a transmembrane channel for facilitated diffusion of protons to the stroma, breaking down the proton gradient.
• CF₁: Protrudes on the outer surface of the thylakoid membrane, where the energy from the gradient breakdown causes a conformational change, enabling ATP synthesis.

• Energy from proton pumping creates a high proton concentration in the thylakoid lumen. ATP synthase channels protons back across the membrane, releasing energy to catalyze ATP formation.

Dark Reaction (Biosynthetic Phase) - Use of ATP & NADPH

• Products of the light reaction are ATP, NADPH, and O₂. The dark reaction uses ATP and NADPH to drive the synthesis of food (sugars).

• This phase does not directly depend on light but relies on light reaction products. When light becomes unavailable, biosynthesis continues briefly before stopping, resuming when light is available.

• CO₂ combines with H₂O to form (CH₂O)ₙ or sugars

• CO₂ assimilation during photosynthesis occurs via two pathways:

• C3 pathway: The first stable product of CO₂ fixation is a C3 acid (3-phosphoglyceric acid - PGA). Discovered by Melvin Calvin using ¹⁴C in algal photosynthesis.
• C4 pathway: The first stable product is oxaloacetic acid (OAA), a 4-carbon organic acid.

C₃ Pathway (Calvin Cycle)

• The Calvin cycle occurs in all photosynthetic plants (C3 or C4 pathways) and has three stages: Carboxylation, Reduction, and Regeneration.

1. Carboxylation of RuBP

• RuBP (ribulose bisphosphate), a 5-carbon ketose sugar, is the primary CO₂ acceptor.

• CO₂ is fixed by RuBP to form two 3-PGA molecules, catalyzed by the enzyme RuBP carboxylase.

• This enzyme, also called RuBP carboxylase-oxygenase (RuBisCO), has oxygenation activity and is the most abundant enzyme globally.

2. Reduction

• A series of reactions leads to glucose formation, using 2 ATP for phosphorylation and 2 NADPH for reduction per CO₂ molecule.

• Fixation of 6 CO₂ molecules requires 6 turns of the cycle to produce one glucose molecule.

3. Regeneration of RuBP

• Crucial for cycle continuation, it requires one ATP for phosphorylation to regenerate RuBP.

• For each CO₂ molecule, 3 ATP and 2 NADPH are needed. Cyclic photophosphorylation may occur to balance ATP and NADPH usage.

• Producing one glucose molecule requires 6 turns of the cycle.

What goes in and comes out of the Calvin cycle?

In	Out
6 CO₂	1 glucose
18 ATP	18 ADP
12 NADPH	12 NADP

C₄ Pathway (Hatch & Slack Pathway)

• Present in plants adapted to dry tropical regions, which also use the C3 pathway as the main biosynthetic pathway.

• Large cells around vascular bundles, called bundle sheath cells, form Kranz anatomy (‘wreath’), with multiple layers, many chloroplasts, thick walls impervious to gas exchange, and no intercellular spaces.

Steps of Hatch and Slack Pathway

• The primary CO₂ acceptor is phosphoenol pyruvate (PEP), a 3-carbon molecule in mesophyll cells, fixed by the enzyme PEP carboxylase (PEPcase).

• Mesophyll cells lack RuBisCO.

• The C4 acid OAA forms in mesophyll cells, converting to malic acid or aspartic acid, which are transported to bundle sheath cells.

• In bundle sheath cells, C4 acids break down to release CO₂ and a C3 molecule, which is transported back to mesophyll cells and converted to PEP.

• Released CO₂ enters the C3 pathway. Bundle sheath cells are rich in RuBisCO but lack PEPcase.

• C4 plants are special due to:

• Kranz anatomy.
• Tolerance to higher temperatures.
• Response to high light intensities.
• Lack of photorespiration.
• Greater biomass productivity.

Photorespiration

• In the Calvin pathway, RuBP combines with CO₂.

• RuBisCO can bind both CO₂ and O₂, with a higher affinity for CO₂. Binding is competitive, determined by O₂ and CO₂ concentrations.

• In C3 plants, some O₂ binds to RuBisCO, reducing CO₂ fixation. RuBP binds O₂ to form one phosphoglycerate and one phosphoglycolate, a process called photorespiration.

• Photorespiration produces no sugars, ATP, or NADPH, and is wasteful, releasing CO₂ using ATP.

• C4 plants avoid photorespiration by increasing CO₂ concentration at the enzyme site when C4 acids break down in bundle sheath cells, minimizing RuBisCO’s oxygenase activity.

• This lack of photorespiration enhances productivity and yields in C4 plants, along with tolerance to higher temperatures.

Differences between C₃ and C₄ Plants

C₃ Plants	C₄ Plants
Photosynthesis occurs in mesophyll cells.	In mesophyll and bundle sheath cells.
Kranz anatomy is absent.	Present.
RuBP is the primary CO₂ acceptor.	PEP is the primary CO₂ acceptor.
3-PGA, a 3-C compound, is the first stable product.	OAA, a 4-C compound, is the first stable product.
Chloroplasts are of one type (granal).	Dimorphic (granal in mesophyll, agranal in bundle sheath).
Photorespiratory loss is high.	Photorespiration is absent or negligible.
High CO₂ compensation point (25-100 μl CO₂ l⁻¹).	Low CO₂ compensation point (0-10 μl CO₂ l⁻¹).
Optimum temperature for photosynthesis is about 25°C.	About 35°C - 45°C.
Photosynthetically less efficient, low productivity.	Photosynthetically more efficient, high productivity.
E.g., rice, wheat, bean, potato.	E.g., maize, sugarcane, amaranth, sorghum.

Factors Affecting Photosynthesis

• Internal (Plant) factors: Number, size, age, and orientation of leaves, mesophyll cells, and chloroplasts, as well as internal CO₂ concentration and the amount of chlorophyll.

Plant factors depend on the genes and growth of plant.

• External factors: Sunlight, temperature, CO₂ concentration, and water.

Blackman’s Law of Limiting Factors (1905)

• “If a biochemical process is affected by more than one factor, its rate is determined by the factor nearest to its minimal value: it is the factor which directly affects the process if its quantity is changed.”

• For example, a plant with green leaves, optimal light, and CO₂ may not photosynthesize at very low temperatures. Providing optimal temperature enables photosynthesis.

Light

• Light quality, light intensity, and duration of exposure influence photosynthesis.

• At low light intensities, there is a linear relationship between incident light and CO₂ fixation rates. At higher intensities, the rate plateaus as other factors become limiting.

• Light saturation occurs at 10% of full sunlight, so light is rarely limiting in nature, except for shade plants or dense forests.

• Excessive light can break down chlorophyll, reducing photosynthesis.

Carbon Dioxide Concentration

• CO₂ is the major limiting factor for photosynthesis, with atmospheric levels at 0.03–0.04%.

• Increasing CO₂ up to 0.05% boosts fixation rates, but higher levels can be damaging over time.

• At low light, C3 and C4 plants show no response to high CO₂. At high light, both show increased photosynthesis rates.

• C4 plants reach saturation at about 360 μl L⁻¹, while C3 plants respond to higher CO₂, saturating beyond 450 μl L⁻¹, indicating current CO₂ levels limit C3 plants.

• C3 plants’ response to elevated CO₂ increases photosynthesis and productivity, utilized in CO₂-enriched greenhouses for crops like tomatoes and bell peppers.

Temperature

• Dark reactions, being enzymatic, are temperature-sensitive, while light reactions are less affected.

• C4 plants respond to higher temperatures with increased photosynthesis rates, while C3 plants have a lower temperature optimum.

• Temperature optimum of plants also depends on their habitat. Tropical plants have a higher temperature optimum than the plants adapted to temperate climates.

Water

• Water stress closes stomata, reducing CO₂ availability.

• Water stress also causes leaf wilting, decreasing surface area and metabolic activity.