Topic 13: Photosynthesis

13.1 Photosynthesis as an energy transfer process

Students should be able to:

1) describe the relationship between the structure of chloroplasts, as shown in diagrams and electron micrographs, and their function

2) explain that energy transferred as ATP and reduced NADP from the light-dependent stage is used during the light independent stage (Calvin cycle) of photosynthesis to produce complex organic molecules

3) state that within a chloroplast, the thylakoids (thylakoid membranes and thylakoid spaces), which occur in stacks called grana, are the site of the light-dependent stage and the stroma is the site of the light-independent stage

4) describe the role of chloroplast pigments (chlorophyll a, chlorophyll b, carotene and xanthophyll) in light absorption in thylakoids

5) interpret absorption spectra of chloroplast pigments and action spectra for photosynthesis

6) describe and use chromatography to separate and identify chloroplast pigments (reference should be made to Rf values in identification of chloroplast pigments)

7) state that cyclic photophosphorylation and non-cyclic photophosphorylation occur during the light-dependent stage of photosynthesis

8) explain that in cyclic photophosphorylation: • only photosystem I (PSI) is involved • photoactivation of chlorophyll occurs • ATP is synthesised

9) explain that in non-cyclic photophosphorylation:

• photosystem I (PSI) and photosystem II (PSII) are both involved

• photoactivation of chlorophyll occurs

• the oxygen-evolving complex catalyses the photolysis of water

• ATP and reduced NADP are synthesised

10) explain that during photophosphorylation:

• energetic electrons release energy as they pass through the electron transport chain

• the released energy is used to transfer protons across the thylakoid membrane

• protons return to the stroma from the thylakoid space by facilitated diffusion through ATP synthase, providing energy for ATP synthesis

11) outline the three main stages of the Calvin cycle:

• rubisco catalyses the fixation of carbon dioxide by combination with a molecule of ribulose bisphosphate (RuBP), a 5C compound, to yield two molecules of glycerate 3-phosphate (GP), a 3C compound

• GP is reduced to triose phosphate (TP) in reactions involving reduced NADP and ATP

• RuBP is regenerated from TP in reactions that use ATP

12) state that Calvin cycle intermediates are used to produce other molecules, limited to GP to produce some amino acids and TP to produce carbohydrates, lipids and amino acids

(1) Describe the relationship between the structure of chloroplasts, as shown in diagrams and electron micrographs, and their function

The chloroplast has a specialised internal structure that is closely tied to its function in photosynthesis. Key structural features and their functional significance include:

Double-membrane envelope: The chloroplast has an outer and inner membrane which encloses the stroma. This envelope helps maintain the internal environment needed for photosynthetic reactions.
Thylakoids: Inside the stroma there are flattened membrane sacs called thylakoids. These often stack to form grana (singular: granum). The thylakoid membranes contain the chlorophyll pigments, photosystems (PS I and PS II), electron transport chain components and ATP synthase. Because the light-dependent reactions take place at membranes, the extensive surface area provided by thylakoids (and grana) is crucial for capturing light energy, facilitating electron flow, and enabling proton gradients.
Thylakoid lumen (spaces inside thylakoids): Protons (H⁺) accumulate in the thylakoid space during the light-dependent reactions, creating a proton gradient. The return of these protons to the stroma through ATP synthase drives ATP formation.
Stroma: The fluid matrix surrounding the thylakoids is the site of the light-independent (Calvin) cycle. It contains the enzymes (e.g., Rubisco), the chloroplast DNA, ribosomes and other components. The stroma allows the products of the light-dependent stage (ATP and NADPH) to diffuse and be used for carbon fixation and synthesis of organic molecules.
Granal-stromal architecture: The close association between grana and stroma allows efficient transfer of products from the light reaction (thylakoid) to the dark reaction (stroma).
Chloroplast positioning and chlorophyll-pigment arrangement: Chloroplasts are arranged in the mesophyll of leaves, often with their thylakoid surfaces aligned to optimise light capture.
Electron micrographs: These show the detailed ultrastructure — many thylakoid membranes, stacked grana, intergranal lamellae linking grana, etc. Understanding these micrographs helps students visualise how structural complexity translates to functional efficiency (i.e., large surface area for light capture and proton pumping).

In summary, the structural features of chloroplasts (e.g., thylakoid membranes, lumen, stroma, granal stacks) are all engineered (by evolution) to maximise the efficiency of converting light energy into chemical energy and then using that chemical energy for synthesising organic molecules.

(2) Explain that energy transferred as ATP and reduced NADP from the light-dependent stage is used during the light-independent stage (Calvin cycle) of photosynthesis to produce complex organic molecules

In the two major stages of photosynthesis (light-dependent and light-independent), there is a transfer of energy and chemical intermediates between stages:

In the light-dependent reactions (occurring in the thylakoid membranes of chloroplasts) light energy is captured by pigments and used to drive the formation of ATP (via photophosphorylation) and to reduce NADP⁺ to NADPH (sometimes written as reduced NADP).
These energy-rich products (ATP and NADPH) are then the energy currency and reducing power for the light-independent reactions (also known as the Calvin cycle) which take place in the stroma of the chloroplast.
During the Calvin cycle, carbon dioxide is fixed and built into organic molecules (initially triose phosphates), and then further converted (or used) to produce carbohydrates, lipids, amino acids and other complex organic molecules. The ATP provides the energy to drive the endergonic steps of fixing CO₂ and regenerating starting compounds; NADPH provides the reducing equivalents (electrons/hydrogens) needed to reduce 3-phosphoglycerate/triose phosphates in the cycle.
Without the ATP and reduced NADP supplied by the light reactions, the Calvin cycle cannot proceed, and no complex organic molecules (e.g., glucose, starch) can be produced. Thus there is a clear relationship: light energy → chemical energy (ATP/NADPH) → carbon fixation and organic molecule synthesis.

(3) State that within a chloroplast, the thylakoids (thylakoid membranes and thylakoid spaces), which occur in stacks called grana, are the site of the light-dependent stage and the stroma is the site of the light-independent stage

This point simply requires the student to state the sites of the two major photosynthetic stages within the chloroplast:

Light-dependent stage: Occurs in the thylakoid membranes and thylakoid spaces (lumen). Specifically, the membrane components (photosystems, electron transport chain, ATP synthase) are located here (in the grana stacks).
Light-independent stage (Calvin cycle): Occurs in the stroma – the fluid filled space surrounding the thylakoids within the chloroplast.

This mapping of structure to stage is important and must be memorised.

(4) Describe the role of chloroplast pigments (chlorophyll a, chlorophyll b, carotene and xanthophyll) in light absorption in thylakoids

Chloroplasts contain a range of pigments which capture light of different wavelengths and transfer that energy to the reaction centres of photosystems. Key points:

Chlorophyll a is the primary pigment in the reaction centres of Photosystem I (PS I) and Photosystem II (PS II). It absorbs mainly red (≈ 680 nm, 700 nm) and blue light, reflecting green, which is why leaves appear green.
Chlorophyll b is an accessory pigment; its absorption spectrum is slightly different (it absorbs light around ≈ 455 nm and 640 nm), thereby widening the range of light that can be harvested. It passes energy to chlorophyll a.
Carotene (a carotenoid) absorbs blue–violet light and reflects orange; xanthophylls (another class of carotenoids) absorb in similar ranges and reflect yellow. These accessory pigments extend the range of wavelengths that the chloroplast can use for photosynthesis, and also help protect the photosystems from photo-oxidation (by dissipating excess energy).
Role in thylakoids: These pigments are embedded in the thylakoid membrane; when they absorb photons, their electrons become excited and this energy is funnelled (via resonance energy transfer) to the reaction centre chlorophyll a, causing photoactivation (electron transfer).
By having multiple pigments, the chloroplast can absorb a broader spectrum of light (not just the red/blue peaks of chlorophyll a), increasing overall efficiency of light harvesting in varying light conditions.
The pigments also help adapt plants to different light environments (shade, bright sun) by varying pigment composition.

Thus, the role of these pigments is to absorb light energy at different wavelengths and transfer that energy to the photosystems where it initiates the light-dependent reactions.

(5) Interpret absorption spectra of chloroplast pigments and action spectra for photosynthesis

Students must be able to read and interpret two types of graphs:

Absorption spectrum: A graph plotting the absorption (y-axis) of a pigment (or mixture of pigments) versus wavelength of light (x-axis). It shows peaks and troughs indicating which wavelengths the pigment absorbs strongly. For example, chlorophyll a has peaks in red and blue regions, with low absorption in green.
Action spectrum: A graph showing the rate (or relative rate) of photosynthesis (y-axis) versus wavelength of light (x-axis). It indicates which wavelengths lead to highest photosynthetic activity. The peaks usually correspond to wavelengths that are strongly absorbed by the pigments.
Interpretation involves:
- Recognising that high absorption wavelengths in the pigment absorption spectrum often align with high photosynthetic rate in the action spectrum (because those wavelengths provide more useful energy).
- Noting differences between the two spectra: for example, accessory pigments may absorb light strongly (absorption spectrum) but may have less visible effect on photosynthetic rate because the transfer to reaction centre may be less efficient or because pigments are less abundant.
- Understanding troughs/low-absorption regions (e.g., green light) correlate with lower rates of photosynthesis (green light is largely reflected, not absorbed).
- Making inferences: e.g., if a plant has a pigment absorbing at 500 nm and the action spectrum peak corresponds, then that pigment is contributing meaningfully.
In an exam context, students might be given graphs and asked to identify peaks, compare spectra, explain why absorption peaks correspond or why action spectrum peaks may not exactly match pigment absorption peaks (because of accessory pigments and transfer efficiencies).

(6) Describe and use chromatography to separate and identify chloroplast pigments (reference should be made to Rf values in identification of chloroplast pigments)

Chromatography (typically paper or thin-layer) is used in practical work to separate the different pigments in chloroplasts. Key points for description and use:

Procedure outline: Pigment extract (from crushed leaves with solvent) is applied as a small spot near one end of chromatography paper or TLC plate. A suitable solvent (mobile phase) is allowed to ascend by capillary action. Pigments travel at different rates depending on their solubility in solvent and their affinity for the stationary phase. The result is a series of separated pigment bands (e.g., carotene, xanthophyll, chlorophyll a, chlorophyll b).
Identification: The separated pigments can be identified by colour (orange for carotene, yellow for xanthophyll, greenish for chlorophylls) and by calculating Rf values for each spot:

Students must be aware that comparing measured Rf values with known values allows identification of each pigment.
Use: Students may carry out an investigation comparing pigments in different plant species, or under different light conditions, to observe differences in pigment composition and Rf values. They must record data, identify pigments, and draw conclusions.
Reference to chloroplast pigments: The chromatography separates exactly the pigments referred earlier (chlorophyll a, chlorophyll b, carotene, xanthophyll). Students should link the separated bands back to their absorption roles.
In summary: chromatography is a practical technique to separate and identify chloroplast pigments; understanding of Rf value calculation and interpretation is required.

(7) State that cyclic photophosphorylation and non-cyclic photophosphorylation occur during the light-dependent stage of photosynthesis

Students should state that during the light-dependent reactions of photosynthesis two pathways for electron flow exist:

Cyclic photophosphorylation: electrons circulate back to the photosystem (usually Photosystem I) and only ATP is produced.
Non-cyclic photophosphorylation: electrons follow a non-returning (linear) path from Photosystem II → electron transport chain → Photosystem I → ultimately to NADP⁺, forming NADPH, and ATP is also produced; water is split (photolysis) and oxygen is released.

This statement simply acknowledges the two pathways.

(8) Explain that in cyclic photophosphorylation:

only photosystem I (PS I) is involved
photoactivation of chlorophyll occurs
ATP is synthesised
In cyclic photophosphorylation, the mechanism specifically involves:
Photosystem I only: Light is absorbed by PSI, exciting electrons which travel through an electron transport chain and return to the same PSI complex. Because PS II is not involved, water is not split and no oxygen is generated.
Photoactivation of chlorophyll: The chlorophyll in PSI absorbs photons; the electrons reach a higher energy level (“excited”). These excited electrons are passed to the electron transport chain, releasing energy as they move back to lower potentials.
ATP synthesis: The energy released by electrons moving through the chain is used to pump protons (H⁺) across the thylakoid membrane (into lumen) generating a proton gradient. Protons flow back via ATP synthase, driving phosphorylation of ADP to ATP. Owing to the cyclic path, the only utilitarian product is ATP (i.e., no NADPH, no O₂).
Additional notes: Because cyclic photophosphorylation only produces ATP, it is useful when the plant has sufficient NADPH but requires extra ATP (for example, for the Calvin cycle). Elasticity in the light reactions is partly achieved via the cyclic route.

(9) Explain that in non-cyclic photophosphorylation:

photosystem I (PS I) and photosystem II (PS II) are both involved
photoactivation of chlorophyll occurs
the oxygen-evolving complex catalyses the photolysis of water
ATP and reduced NADP are synthesised
Non-cyclic photophosphorylation involves the linear flow of electrons and results in both ATP and NADPH production (and oxygen release). Explanation details:
PS I and PS II involvement: Light is absorbed by both PS II and PS I. Electrons are excited in PS II, passed down an electron transport chain to PS I, re-excited, and finally passed to NADP⁺ to form NADPH.
Photoactivation of chlorophyll: In both photosystems, chlorophyll molecules absorb photons; their electrons move to higher energy states and are captured by primary electron acceptors.
Oxygen-evolving complex and photolysis of water: PS II has an oxygen-evolving complex (also called water-splitting complex) that oxidises water: 2H₂O → 4H⁺ + 4e⁻ + O₂. The electrons supplied here replace those lost by PS II when they are excited and passed down the chain; protons contribute to the proton gradient; O₂ is released as a by-product into the atmosphere.
ATP and reduced NADP (NADPH) synthesised: As electrons travel through the electron transport chain between PS II and PS I, energy is released; this energy is used to pump protons into the lumen, generating a proton gradient. Protons flow back through ATP synthase to generate ATP. Simultaneously, at PS I the electrons reduce NADP⁺ + H⁺ → NADPH. Therefore, the plant obtains both ATP and reducing power (NADPH) needed for the Calvin cycle.
Significance: Non-cyclic photophosphorylation is the main pathway under normal photosynthetic conditions, enabling full conversion of light energy into chemical energy, carbon fixation and ultimately synthesis of organic molecules.

(10) explain that during photophosphorylation:

In the light-dependent stage of photosynthesis (occurring in the thylakoid membranes of the chloroplast), a process called photophosphorylation occurs. The term means “using light (photo) to add phosphate (phosphorylation) to ADP → ATP”. Here is how the three bullet-points are fulfilled:

Energetic electrons release energy as they pass through the electron transport chain
- When light hits the pigment molecules (chlorophyll etc) in the photosystems, electrons become excited to a higher energy state.
- These excited (energetic) electrons are then transferred from one carrier molecule to another along the thylakoid membrane (i.e., an electron transport chain). For example, electrons from photosystem II (PS II) through a sequence of redox steps, and then to photosystem I (PS I).
- As the electrons move (fall) from higher energy to lower energy states through the chain, they release energy. That is, the electrons’ potential energy is converted into usable energy in the process.
The released energy is used to transfer protons across the thylakoid membrane
- The energy released by the electrons is harnessed to actively pump protons (H⁺ ions) from one side of the thylakoid membrane to the other — specifically from the stroma (outside of the thylakoid lumen) into the thylakoid space/lumen.
- This movement builds up a high concentration of H⁺ inside the thylakoid lumen relative to the stroma, creating an electrochemical (proton) gradient (sometimes called the proton-motive force).
Protons return to the stroma from the thylakoid space by facilitated diffusion through ATP synthase, providing energy for ATP synthesis
- Once a proton gradient is in place (many H⁺ inside the lumen, fewer in the stroma), protons flow back down their gradient through specialised channels in the membrane (ATP synthase). This is facilitated diffusion (passive movement via a protein channel).
- The flow of protons releases energy (as they move down the gradient) and that energy is used to drive the phosphorylation of ADP + Pi → ATP. In other words, ATP synthesis is powered by the returning protons.
- Because the whole process started with light energy (which excited the electrons), and ends with ATP produced, it is described as photophosphorylation.

(11) Outline the three main stages of the Calvin cycle:

fixation by rubisco of carbon dioxide by combination with ribulose bisphosphate (RuBP), a 5C compound, to yield two molecules of GP (PGA), a 3C compound
the reduction of GP to triose phosphate (TP) involving ATP and reduced NADP
the regeneration of ribulose bisphosphate (RuBP) using ATP
The Calvin cycle (light-independent stage) can be outlined in three major sequential phases:

Carbon fixation – The enzyme RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyses the reaction: CO₂ + ribulose-1,5-bisphosphate (RuBP, 5-carbon compound) → two molecules of glycerate-3-phosphate (GP or PGA; 3-carbon each).
Reduction – The GP molecules are phosphorylated by ATP and then reduced by NADPH (i.e., reduced NADP) to form triose phosphate (TP; 3-carbon). The formula might be simplified as: GP + ATP + NADPH → TP + ADP + Pi + NADP⁺.
Regeneration of RuBP – Some of the TP (or a derivative thereof) is used, via ATP-dependent reactions, to regenerate RuBP so the cycle can continue. The net result: for each 3 CO₂ fixed, five molecules of TP are used to regenerate three molecules of RuBP, and one molecule of TP is left for biosynthesis (or gluconeogenesis).
Additional notes: The TP (triose phosphate) can then be converted into carbohydrates (e.g., glucose, starch), lipids or amino acids (via intermediary metabolism) depending on the cell’s needs. The ATP and NADPH used come from the light-dependent stage.
In summary, the Calvin cycle uses chemical energy (ATP) and reducing power (NADPH) to convert atmospheric CO₂ into organic carbon compounds that the plant can use for growth and storage.

(12) Describe, in outline, the conversion of Calvin cycle intermediates to carbohydrates, lipids and amino acids and their uses in the plant cell

After the Calvin cycle produces triose phosphates (TP), these intermediates can be channelled into various biosynthetic pathways:

Carbohydrates: Triose phosphate molecules can be converted into hexose phosphates (e.g., glucose-6-phosphate) and then polymerised into starch (in plastids) for storage, or transported as sucrose (in phloem) to other parts of the plant. Carbohydrates provide energy reserves and structural components (e.g., cellulose).
Lipids (fats/oils): Some of the TP or other intermediates (e.g., glycerate, glycerol-3-phosphate) can be used as precursors for fatty acid synthesis. Fatty acids combine with glycerol to form triacylglycerols (storage lipids) or phospholipids (membrane lipids). Lipids are energy-rich and have structural roles (membranes).
Amino acids: Organic carbon skeletons from Calvin cycle intermediates (e.g., 3-carbon or 5-/6-carbon sugars) can combine with nitrogen (from nitrate/ammonium uptake) to form amino acids via transamination and other reactions. Amino acids are then used to synthesise proteins, enzymes and other nitrogen-containing compounds.
Uses in the plant cell:
- Energy supply: stored carbohydrates and lipids can be mobilised when needed (respiration).
- Structural: cellulose (from carbohydrate) builds cell walls; phospholipids form membranes.
- Metabolic: enzymes and structural proteins made from amino acids catalyse and regulate cellular reactions; some lipids act as signalling molecules.
Thus the Calvin cycle is not only about producing simple sugars but also about generating building blocks for a wide range of vital biomolecules in the plant.

13.2 Investigation of limiting factors

Students should be able to:

1) state that light intensity, carbon dioxide concentration and temperature are examples of limiting factors of photosynthesis 2) explain the effects of changes in light intensity, carbon dioxide concentration and temperature on the rate of photosynthesis

3) describe and carry out investigations using redox indicators, including DCPIP and methylene blue, and a suspension of chloroplasts to determine the effects of light intensity and light wavelength on the rate of photosynthesis

4) describe and carry out investigations using whole plants, including aquatic plants, to determine the effects of light intensity, carbon dioxide concentration and temperature on the rate of photosynthesis

(1) (From limiting factors) Explain the term limiting factor in relation to photosynthesis

A limiting factor is a condition or resource whose quantity or level restricts the rate of a biological process (in this case, photosynthesis) when other factors are in sufficient supply. If one factor is in inadequate supply, then increasing the supply of other factors will not increase the rate of the process until that limiting factor is increased. In context:

For photosynthesis, the main potential limiting factors are: light intensity, carbon dioxide concentration, temperature (and sometimes chlorophyll or water availability).
Example: If CO₂ concentration is very low, then even if light intensity is high and temperature is ideal, photosynthesis cannot increase further until CO₂ increases. At that point, CO₂ is the limiting factor. If light intensity becomes increased further, but CO₂ is still low, no further increase will occur because CO₂ is the limiting factor.
In practice, for leaves or crop plants, at different times different factors may become limiting (e.g., in a greenhouse at midday, CO₂ might be limiting; at dusk, light might be limiting).
Understanding limiting factors is crucial for optimising plant growth (e.g., in horticulture or glasshouses) because one can manipulate the factor that is limiting to boost overall rate.

(2) Explain the effects of changes in light intensity, carbon dioxide concentration and temperature on the rate of photosynthesis

The rate of photosynthesis depends on several environmental factors. Each of the three–light intensity, CO₂ concentration and temperature–affects the process in characteristic ways:

Light intensity: As light intensity increases, the light-dependent reactions (absorption of photons, photolysis of water, electron transport, ATP and NADPH production) speed up, which means more ATP/NADPH for the Calvin cycle → higher rate of carbon fixation → net photosynthesis increases. However, above a certain light intensity the rate plateaus because another factor becomes limiting (e.g., CO₂, enzyme capacity). At very high intensities the rate may drop due to photoinhibition.
Carbon dioxide concentration: CO₂ is the substrate for the Calvin cycle (carbon fixation). At low CO₂, the Calvin cycle cannot fix more carbon even if ATP/NADPH are abundant. As CO₂ concentration increases, the rate rises until something else becomes limiting (light, temperature, enzyme availability). At very high CO₂ it may plateau.
Temperature: Photosynthesis involves enzyme-mediated reactions (e.g., Rubisco) and diffusion processes. At low temperatures, kinetic energy is low and the rate of enzyme activity is slow → lower photosynthetic rate. As temperature increases, the rate increases (up to the optimum for those enzymes). Beyond the optimum, enzyme denaturation, membrane fluidity changes and gas solubility changes (i.e., O₂ vs CO₂) can cause the rate to decrease. If temperature is too high, enzymes may become denatured and the rate drops sharply.
Interaction between factors: Often the rate is determined by whichever factor is limiting at a given moment. For example:
- At low light, increasing CO₂ may have little effect (light is limiting).
- At moderate high light and temperature, increasing CO₂ may increase the rate (CO₂ becomes limiting).
- At very high temperature, temperature may limit.
Practical significance: Understanding these effects is important in agriculture (e.g., glasshouse growth, shading, CO₂ enrichment, temperature control) to maximise yield.

(3) Carry out an investigation to determine the effect of light intensity or light wavelength on the rate of photosynthesis (using a redox indicator, e.g., DCPIP, and a suspension of chloroplasts)

Students need to be familiar with practical investigations in which the effect of light intensity or wavelength on photosynthesis is measured via a redox indicator such as DCPIP:

Concept: DCPIP (2,6-dichlorophenol-indophenol) is blue in its oxidised form and becomes colourless when reduced. In a chloroplast suspension under light, the electrons from the light-dependent reactions reduce DCPIP instead of NADP⁺ (when NADP⁺ is omitted). As the rate of DCPIP decolourisation is proportional to the rate of the light reaction (electron flow), it is used as a proxy for photosynthesis rate.
Procedure (outline): Isolate chloroplasts (or a thylakoid suspension) from leaves; set up into cuvettes/tubes with DCPIP; vary the light intensity (by varying distance from light source) or use filters to allow different wavelengths; start the reaction and record the time taken for the DCPIP to lose colour or measure absorbance decline at known wavelength. Control experiments (in the dark, or with broken chloroplasts) show negligible colour change.
Variables: Independent variable: light intensity (distance from lamp, shading) or wavelength (colour filters). Dependent variable: rate of DCPIP reduction (time taken, absorbance change). Control variables: chloroplast concentration, volume, temperature, light source, DCPIP concentration.
Interpretation: Plot rate vs light intensity or wavelength; expect increasing rate with increasing light until plateau; expect maxima for particular wavelengths corresponding to pigment absorption peaks; minima for non-absorbed wavelengths (e.g., green light).
Limitations: The method measures electron flow not full carbon fixation; chloroplast isolation may damage membranes; filters may change intensity; temperature fluctuations can influence rate. Students should evaluate these when interpreting results.
This investigation helps students understand how light affects the light-dependent reactions and provides hands-on link between theory (pigment absorption, photophosphorylation) and experiment.

(4) Carry out investigations on the effects of light intensity, carbon dioxide and temperature on the rate of photosynthesis using whole plants, e.g., aquatic plants such as Elodea and Cabomba

Students must also be able to describe and carry out investigations using whole (living) plants rather than just chloroplast extracts. For example, using aquatic plants that release oxygen bubbles as a measure of photosynthesis. Key details:

Apparatus/Setup: Use an aquatic plant (Elodea or Cabomba) in a water bath with a known concentration of CO₂ (which may be controlled by adding sodium hydrogencarbonate). Illuminate the plant with a light source at varying intensities, or change CO₂ concentration or temperature. Measure rate of photosynthesis via number of oxygen bubbles released per unit time, or volume of oxygen collected under an inverted funnel or gas syringe.
Independent variables: Light intensity (distance from lamp, shading), CO₂ concentration (via bicarbonate addition or CO₂ removal), temperature (using water bath at different temps).
Dependent variable: Rate of oxygen production (bubbles/time, ml O₂ per minute, etc.).
Control variables: Plant species, light duration, water volume, initial CO₂ concentration, temperature (when not the variable), time measurement, plant size/health, same lamp type.
Interpretation: Typically, increasing light intensity or CO₂ up to a point increases oxygen output; beyond that a plateau or decline may occur (limiting factor changes). Increasing temperature boosts metabolic/enzymatic activity up to an optimum; beyond that the rate decreases due to denaturation or stress.
Practical considerations: Ensure sufficient acclimatisation of plant, uniform plant size, avoid heating the water too much from lamp, use consistent light wavelength, ensure enough time for equilibrium. Graduated cylinders or gas syringes must be calibrated.
Data handling: Rate graphs (oxygen output vs independent variable), identification of optimum, quantification of effect. Students should also discuss reliability, repeats, possible error sources (e.g., bubble coalescence, plant fatigue).
This practical links the theoretical limiting‐factor concepts with real‐life whole-plant responses, and gives hands-on experience in experimental design and interpretation.

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