Photosynthesis in higher plants:

Perspectives in Biological Study and Unit Overview

• Two Historical Perspectives on Biology:

  • Organismic Level and Above: This perspective focuses on structure and variation of living organisms over time and resulted in the discipline of ecology and related fields.

  • Cellular and Molecular level: This perspective focuses on the internal mechanics of life, leading to the development of physiology and biochemistry.

• Scope of Plant Physiology:

  • The unit focuses on physiological processes in flowering plants.

  • Key processes described include photosynthesis, respiration, and plant growth and development.

  • Descriptions are provided in molecular terms within the context of cellular activities and the organism level.

  • Relations between physiological processes and the environment are discussed where appropriate.

Melvin Calvin and the Mapping of Carbon Assimilation

• Biographical Details:

  • Born in Minnesota in April 1911.

  • Received his Ph.D. in Chemistry from the University of Minnesota.

  • Served as a Professor of Chemistry at the University of California, Berkeley.

• Scientific Contribution:

  • Following World War II, Calvin and his co-workers sought beneficial uses for radioactivity in response to the Hiroshima-Nagasaki bombings.

  • Methodology: Along with J.A. Bassham, he studied reactions in green plants that form sugars from raw materials ($CO_2$, water, and minerals) by labeling carbon dioxide with the radioactive isotope ${^{14}C}$.

  • Propositions: Calvin proposed that plants convert light energy into chemical energy by transferring an electron in an organized array of pigment molecules.

  • The Calvin Cycle: He mapped the complete pathway of carbon assimilation in photosynthesis.

• Recognition and Legacy:

  • Awarded the Nobel Prize in 1961.

  • His principles of photosynthesis are currently used in research for renewable energy, materials, and basic solar energy studies.

Introduction to Photosynthesis

• Basic Definition: Photosynthesis is a physico-chemical process by which green plants use light energy to drive the synthesis of organic compounds.

• Autotrophs vs. Heterotrophs:

  • Autotrophs: Green plants that synthesize the food they need.

  • Heterotrophs: All other organisms (including humans) that depend on green plants for food.

• Importance of Photosynthesis:

  • It is the primary source of all food on earth.

  • It is responsible for the release of oxygen ($O_2$) into the atmosphere.

• Fundamental Requirements: Early experiments have established that chlorophyll (green pigment), light, and $CO_2$ are essential for the process.

Early Historical Experiments in Photosynthesis

• Joseph Priestley (1733–1804):

  • Performed experiments in 1770 revealing the essential role of air in plant growth.

  • Discovered oxygen in 1774.

  • The Bell Jar Experiment: Observed that a burning candle or a mouse in a closed bell jar would "damage" the air (extinguish the flame or suffocate the mouse). However, when a mint plant was added, the jar supported both life and combustion.

  • Hypothesis: Plants restore to the air whatever breathing animals and burning candles remove.

• Jan Ingenhousz (1730–1799):

  • Demonstrated that sunlight is essential for the process that purifies the air.

  • Aquatic Plant Experiment: Showed that in bright sunlight, small bubbles (later identified as oxygen) formed around the green parts of the plant. In the dark, no bubbles formed.

  • Conclusion: Only the green parts of plants release oxygen.

• Julius von Sachs (1854):

  • Provided evidence that plants produce glucose during growth, usually stored as starch.

  • Identified that chlorophyll is located in special bodies (later called chloroplasts).

  • Conclusion: Green parts are the site of glucose production.

• T.W. Engelmann (1843–1909):

  • Used a prism to split light into a spectrum to illuminate green alga (Cladophora) in a suspension of aerobic bacteria.

  • Observation: Bacteria (used to detect $O_2$ evolution) accumulated mainly in the blue and red light regions.

  • Result: Described the first action spectrum of photosynthesis, which resembles the absorption spectra of chlorophyll a and b.

• Cornelius van Niel (1897–1985):

  • Microbiologist who studied purple and green sulfur bacteria.

  • Demonstration: Photosynthesis is a light-dependent reaction where hydrogen from an oxidizable compound reduces carbon dioxide to carbohydrates.

  • Inference: In green plants, $H_2O$ is the hydrogen donor and is oxidized to $O_2$. For sulfur bacteria, $H_2S$ is the donor, and the product is sulfur or sulfate. This proved oxygen comes from water, not $CO_2$.

  • The Correct General Equation:     $6CO_2 + 12H_2O \xrightarrow{\text{Light}} C_6H_{12}O_6 + 6H_2O + 6O_2$

The Site and Machinery of Photosynthesis

• Location: Occurs primarily in the leaves (mesophyll cells) and other green parts of the plant.

• Chloroplast Structure:

  • Membranous System: Includes grana and stroma lamellae.

  • Matrix: The fluid stroma.

• Division of Labour:

  • Membrane System: Responsible for trapping light energy and synthesizing $ATP$ and $NADPH$. These are the "Light Reactions" or photochemical reactions.

  • Stroma: Site of enzymatic reactions that synthesize sugar (which forms starch). These are the "Dark Reactions" or carbon reactions.

  • Note: Dark reactions are not independent of light; they depend on the products of the light reactions ($ATP$ and $NADPH$).

Photosynthetic Pigments

• Types of Pigments (Separated via Chromatography):

  1. Chlorophyll a: Bright or blue-green; the chief pigment associated with photosynthesis.

  2. Chlorophyll b: Yellow-green.

  3. Xanthophylls: Yellow.

  4. Carotenoids: Yellow to yellow-orange.

• Function of Pigments:

  • Ability to absorb light at specific wavelengths.

  • Chlorophyll a: Shows maximum absorption in the blue and red regions. The action spectrum of photosynthesis correlates with these absorption peaks.

  • Accessory Pigments (Chlorophyll b, Xanthophylls, Carotenoids): Absorb light and transfer energy to chlorophyll a. They enable a wider range of wavelengths to be used and protect chlorophyll a from photo-oxidation.

The Light Reaction (Photochemical Phase)

• Components: Light absorption, water splitting, oxygen release, and formation of intermediate chemical products ($ATP$ and $NADPH$).

• Light Harvesting Complexes (LHC):

  • Pigments are organized into two discrete complexes: Photosystem I ($PS\,I$) and Photosystem II ($PS\,II$).

  • Each system consists of hundreds of pigment molecules ("antennae") bound to proteins.

  • Reaction Centre: A single molecule of chlorophyll a.

    • In $PS\,I$, its absorption peak is at $700\,nm$ ($P700$).

    • In $PS\,II$, its absorption peak is at $680\,nm$ ($P680$).

Electron Transport and the Z-Scheme

• Sequence of Events:

  1. $PS\,II$ ($P680$) absorbs red light, exciting electrons that jump to an orbit farther from the nucleus.

  2. An electron acceptor picks up these electrons and passes them to an Electron Transport System ($ETS$) consisting of cytochromes.

  3. This "downhill" movement is based on a redox potential scale.

  4. Electrons are passed to the pigments of $PS\,I$.

  5. Simultaneously, $PS\,I$ electrons are excited by $700\,nm$ light and passed to another acceptor molecule with greater redox potential.

  6. Electrons move downhill again to $NADP^+$, reducing it to $NADPH + H^+$.

• Z-Scheme: The characteristic shape formed when all carriers are placed in sequence on a redox potential scale.

• Splitting of Water:

  • Electrons lost from $PS\,II$ are replaced by the splitting of water.

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

  • The water-splitting complex is associated with $PS\,II$ on the inner side of the thylakoid membrane.

Photophosphorylation and Chemiosmosis

• Definition: Phosphorylation is the synthesis of $ATP$ from $ADP$ and inorganic phosphate. Photophosphorylation occurs in the presence of light.

• Non-cyclic Photophosphorylation: Occurs when both $PS\,II$ and $PS\,I$ work in series. Results in the synthesis of both $ATP$ and $NADPH + H^+$.

• Cyclic Photophosphorylation:

  • Occurs when only $PS\,I$ is functional.

  • Electrons are cycled back to $PS\,I$ through the $ETS$.

  • Synthesizes only $ATP$ (no $NADPH + H^+$).

  • Location: Stroma lamellae (which lack $PS\,II$ and $NADP$ reductase).

  • Occurs when only wavelengths beyond $680\,nm$ are available.

• Chemiosmotic Hypothesis (Mechanism of ATP Synthesis):

  • ATP synthesis is linked to a proton gradient across the thylakoid membrane.

  • Proton Accumulation in the Lumen:

    1. Water splitting occurs on the inner side of the membrane, releasing protons into the lumen.

    2. As electrons move through the systems, the primary acceptor (on the outer side) transfers electrons to an $H$ carrier. This carrier removes a proton from the stroma and releases it into the lumen.

    3. The $NADP$ reductase enzyme (on the stroma side) removes protons from the stroma to reduce $NADP^+$ to $NADPH + H^+$.

  • Gradient Breakdown: The gradient is broken as protons move through the transmembrane channel of the $CF_0$ portion of the $ATP$ synthase into the stroma.

  • ATP Synthase: Consists of $CF_0$ (embedded channel) and $CF_1$ (protrudes on the stroma side). The movement of protons causes a conformational change in $CF_1$ that catalyzes $ATP$ formation.

The Biosynthetic Phase (The Calvin Cycle)

• General Context: Also known as the "Dark Reaction." It uses $ATP$ and $NADPH$ to fix $CO_2$ into sugars.

• Melvin Calvin's Discovery: Used radioactive $^{14}C$ in algal studies to find the first product: a 3-carbon acid, 3-phosphoglyceric acid ($PGA$).

• Pathways based on First Products:

  • $C_3$ Pathway: First stable product is $PGA$ (3-carbon).

  • $C_4$ Pathway: First stable product is oxaloacetic acid ($OAA$, 4-carbon).

• Stages of the Calvin Cycle ($C_3$):

  1. Carboxylation: $CO_2$ is fixed into a stable intermediate. $CO_2$ combines with the 5-carbon sugar ribulose-1,5-bisphosphate ($RuBP$). Catalyzed by $RuBisCO$ ($RuBP$ carboxylase-oxygenase). Produces two molecules of $3-PGA$.

  2. Reduction: A series of reactions using $2$ molecules of $ATP$ (for phosphorylation) and $2$ of $NADPH$ (for reduction) per $CO_2$ fixed to form glucose.

  3. Regeneration: $RuBP$ is regenerated to continue the cycle, requiring $1\,ATP$.

• Cycle Budget for 1 Glucose Molecule:

  • Requires $6$ turns of the cycle.

  • Input: $6\,CO_2$, $18\,ATP$, $12\,NADPH$.

  • Output: $1\,Glucose$, $18\,ADP$, $12\,NADP$.

The C4 Pathway (Hatch and Slack Pathway)

• Characteristics of $C_4$ Plants: Adapted to dry tropical regions. Features "Kranz" anatomy (large bundle sheath cells forming a wreath around vascular bundles). They lack photorespiration and tolerate high temperatures.

• Anatomy:

  • Bundle Sheath Cells: Thick walls, no intercellular spaces, impervious to gas exchange, many chloroplasts.

  • Evolutionary Advantage: Greater productivity and biomass yield.

• The Mechanism:

  1. Primary $CO_2$ acceptor is phosphoenol pyruvate ($PEP$, 3-carbon) in mesophyll cells.

  2. Fixed by $PEP$ carboxylase ($PEPcase$); mesophyll cells lack $RuBisCO$.

  3. $OAA$ (4-carbon) is formed, then converted to malic or aspartic acid.

  4. These are transported to bundle sheath cells, where they are broken down to release $CO_2$ and a 3-carbon molecule.

  5. The 3-carbon molecule is sent back to mesophyll to regenerate $PEP$.

  6. The released $CO_2$ enters the Calvin cycle ($C_3$) in the bundle sheath cells, which are rich in $RuBisCO$.

Photorespiration

• Mechanism: $RuBisCO$ is the most abundant enzyme and has a dual affinity for $CO_2$ and $O_2$. Binding is competitive and concentration-dependent.

• In $C_3$ Plants: Some $O_2$ binds to $RuBisCO$, causing $RuBP$ to form one molecule of phosphoglycerate ($3C$) and one of phosphoglycolate ($2C$). This pathway releases $CO_2$ and consumes $ATP$, but produces no sugar or $ATP/NADPH$.

• In $C_4$ Plants: Photorespiration does not occur because they increase the internal concentration of $CO_2$ at the $RuBisCO$ site through the transport of $C_4$ acids, ensuring $RuBisCO$ acts as a carboxylase.

Factors Affecting Photosynthesis

• Blackman’s Law of Limiting Factors (1905): If a process is affected by more than one factor, the rate is determined by the factor nearest its minimal value.

• Internal Factors: Genetic predisposition, number/size/age/orientation of leaves, mesophyll cells, chloroplasts, internal $CO_2$ concentration, and chlorophyll amount.

• External Factors:

  • Light: Includes quality, intensity, and duration. Relationship between light and $CO_2$ fixation is linear at low intensities. Saturation occurs at $10\%$ of full sunlight. Very high intensity can cause chlorophyll breakdown.

  • Carbon Dioxide: Major limiting factor. Atmospheric concentration is $0.03\%$–$0.04\%$. Increase up to $0.05\%$ can increase fixation.

    • $C_4$ plants saturate at $360\,\mu lL^{-1}$.

    • $C_3$ plants saturate beyond $450\,\mu lL^{-1}$. Current levels are limiting for $C_3$ plants.

  • Temperature: Dark reactions are enzymatic and highly temperature-controlled. $C_4$ plants have higher temperature optima; tropical plants have higher optima than temperate plants.

  • Water: Stress causes stomatal closure (reducing $CO_2$) and leaf wilting (reducing surface area and metabolic activity).