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OCR A Level Biology Your notes

5.6 Photosynthesis
Contents
5.6.1 Photosynthesis & Respiration
5.6.2 Chloroplast Structure & Function
5.6.3 Photosynthetic Pigments
5.6.4 Practical: Investigating Photosynthetic Pigments with Chromatography
5.6.5 The Light-Dependent Stage
5.6.6 Using the Products of the Light-Dependent Reaction
5.6.7 The Light-Independent Stage
5.6.8 Uses of Triose Phosphate
5.6.9 Factors Affecting the Rate of Photosynthesis
5.6.10 Practical: Investigating Factors Affecting the Rate of Photosynthesis

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5.6.1 Photosynthesis & Respiration

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Photosynthesis & Respiration
Photosynthesis occurs in autotrophic organisms such as plants, algae and cyanobacteria
In the process of photosynthesis, light energy is trapped and used to convert simple inorganic
compounds into complex organic compounds. Energy is stored within these organic compounds
Respiration occurs in all living organisms
Respiration is the process by which energy is released from organic molecules in living cells. The
process can be aerobic (using oxygen) or anaerobic (without using oxygen)
There are several similarities and differences between photosynthesis and the two types of respiration
For example, the coenzyme NADP is used in photosynthesis whereas the coenzyme NAD is used in both
aerobic and anaerobic respiration
Comparing Photosynthesis & Respiration Table

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Your notes

Exam Tip
Remember, energy is never created or destroyed; it is converted from one form to another!

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5.6.2 Chloroplast Structure & Function

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Chloroplast Structure & Function
Chloroplasts are the organelles in plant cells where photosynthesis occurs
These organelles are roughly 2 - 10 μm in diameter (they are larger than mitochondria)
Each chloroplast is surrounded by a double-membrane envelope
Each of the envelope membranes is a phospholipid bilayer
The outer membrane is permeable to a range of ions and small molecules
The inner membrane contains transport proteins that only allow certain molecules or ions to enter
or leave the chloroplast
Chloroplasts are filled with a cytosol-like fluid known as the stroma
CO2, sugars, enzymes and other molecules are dissolved in the stroma
A separate system of membranes is found in the stroma
This membrane system consists of a series of flattened fluid-filled sacs known as thylakoids
The thylakoid membranes contain pigments, enzymes and electron carriers
These thylakoids stack up to form structures known as grana (singular – granum)
Grana are connected by membranous channels called stroma lamellae, which ensure the stacks of
sacs are connected but distanced from each other
The membranes of the grana create a large surface area
This membrane system provides a large number of pigment molecules that ensure as much light
as necessary is absorbed
The pigment molecules are arranged in light-harvesting clusters known as photosystems
In a photosystem, the different pigment molecules are arranged in funnel-like structures in the
thylakoid membrane (each pigment molecule passes energy down to the next pigment molecule
in the cluster until it reaches the primary pigment reaction centre)
The stroma also contains small (70S) ribosomes, a loop of DNA and starch grains:
The loop of DNA codes for some of the chloroplast proteins (other chloroplast proteins are coded
for by the DNA in the plant cell nucleus)
The proteins coded for by this loop of chloroplast DNA are produced at the 70S ribosomes
Sugars formed during photosynthesis are stored as starch inside starch grains

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Your notes

An example of a photosystem in a chloroplast thylakoid membrane: a light-harvesting cluster of

photosynthetic pigments involved in the light-dependent stage of photosynthesis

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Location of the stages of photosynthesis

The process of photosynthesis is made up of two stages, the light-dependent stage and the light- Your notes
independent stage
These stages take place in specific locations within the chloroplast
The first stage, the light-dependent stage takes place on the thylakoid membranes of the grana
Light becomes trapped within the reaction centres of the grana
The light-independent stage takes place in the stroma

The location of the different stages of photosynthesis. The products of the light-dependent reaction
are used in the light-independent reaction.
Adaptations of chloroplasts to photosynthesis
Chloroplasts are specialised organelles that are adapted to carry out photosynthesis

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Stroma: The gel-like fluid contains enzymes that catalyse the reactions of the light-independent
stage. The stroma surrounds the grana and membranes, making the transport of products from the
light-dependent stage into the stroma rapid Your notes
Grana: The granal stacks create a large surface area for the presence of many photosystems which
allows for the maximum absorption of light. It also provides more membrane space for electron
carriers and ATP synthase enzymes
DNA: The chloroplast DNA (cpDNA) contains genes that code for some of the proteins and enzymes
used in photosynthesis
Ribosomes: The presence of ribosomes allows for the translation of proteins coded by cpDNA
Inner membrane of chloroplast envelope: The selective transport proteins present in the inner
membrane control the flow of molecules between the stroma and cytosol (the cytoplasm of the plant
cell)

Exam Tip
Make sure you can identify the structures of a chloroplast on a diagram AND that you can explain the
function of each of these structures.

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5.6.3 Photosynthetic Pigments

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Photosynthetic Pigments
Chloroplasts contain several different photosynthetic pigments within the thylakoids, which absorb
different wavelengths of light
There are two types of pigments: chlorophylls and carotenoids
Accessory pigments that surround the primary pigment absorb both similar and different
wavelengths of light to chlorophyll, this expands the wavelength range that can be absorbed
from light for use in photosynthesis
Chloroplast Pigments Table

Pigment group Name of pigment Colour of pigment

Chlorophyll a Blue-green
Chlorophylls
Chlorophyll b Yellow-green

β carotene Orange
Carotenoids
Xanthophyll Yellow

Chlorophylls absorb wavelengths in the blue-violet and red regions of the light spectrum
They reflect green light, causing plants to appear green
Carotenoids absorb wavelengths of light mainly in the blue-violet region of the spectrum

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Your notes

Chlorophyll and carotenoids absorb light across the visible light spectrum to use in the light-dependent
reaction of photosynthesis
Pigments and photosystems
Within chloroplasts thylakoids stack up to form structures known as grana (singular – granum)
The thylakoid membrane system provides a large number of pigment molecules in an arrangement that
ensures as much light as necessary is absorbed
The pigment molecules are arranged in light-harvesting clusters known as photosystems
In a photosystem, the different pigment molecules are arranged in funnel-like structures in the
thylakoid membrane (each pigment molecule passes energy down to the next pigment molecule in the
cluster until it reaches the primary pigment reaction centre)
There are two different photosystems, each with a specific form of chlorophyll a
Photosystem 1 (PSI), often referred to as P700
The chlorophyll a in this system has a maximum absorption of light at 700nm

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Photosystem 2 (PSII), often referred to as P680

The chlorophyll a in this system has a maximum absorption of light at 680nm
Your notes

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An example of a photosystem in a chloroplast thylakoid membrane: a light-harvesting cluster of

photosynthetic pigments involved in the light-dependent stage of photosynthesis
Your notes
Exam Tip
Remember – the pigments themselves have colour (as described in the table). This is different from the
colours of light that they absorb.

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5.6.4 Practical: Investigating Photosynthetic Pigments with

Chromatography Your notes

Practical: Investigating Photosynthetic Pigments with

Chromatography
Chloroplasts contain several different photosynthetic pigments within the thylakoids, which absorb
different wavelengths of light
Chromatography can be used to separate and identify chloroplast pigments that have been
extracted from a leaf
Chloroplast pigments table

Chromatography
Chromatography is an experimental technique that is used to separate mixtures:
Different components within the mixture travel through the material at different speeds due to
their size and charge
This causes the different components to separate
A retardation factor (Rf) can be calculated for each component of the mixture
Two of the most common techniques for separating these photosynthetic pigments are:
Paper chromatography – the mixture of pigments is passed through paper (cellulose)
Thin-layer chromatography – the mixture of pigments is passed through a thin layer of adsorbent
(eg. silica gel), through which the mixture travels faster and separates more distinctly
Apparatus

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Leaf sample
Distilled water
Pestle and mortar Your notes
Filter paper
Capillary tube
Chromatography solvent
Acetone
Pencil
Ruler
Method
Draw a straight line in pencil approximately 1cm above the bottom of the filter paper being used
Do not use a pen as the ink will separate into pigments within the experiment and obscure the
results
Cut a section of leaf and place it in a mortar
It is important to choose a healthy leaf that has been in direct sunlight so you can be sure it
contains many active photosystems
Add 20 drops of acetone and use the pestle to grind up the leaf sample and release the pigments
Acetone is an organic solvent and therefore fats, such as the lipid membrane, dissolve in it
Acetone and mechanical pressure are used to break down the cell, chloroplast and thylakoid
membranes to release the pigments
Extract some of the pigment using a capillary tube and spot it onto the centre of the pencil line you
have drawn
Suspend the paper in the chromatography solvent so that the level of the solvent is below the pencil
line and leave the paper until the solvent has reached the top of the paper
The mixture is dissolved in the solvent (called the mobile phase) and the dissolved mixture then
passes through a static material (called the stationary phase)
Remove the paper from the solvent and draw a pencil line marking where the solvent moved up to
The pigment should have separated out and there should be different spots on the paper at
different heights above the pencil line, these are the separate pigments
Calculate the Rf value for each spot
Rf value = distance travelled by component (pigment) ÷ distance travelled by the solvent
Always measure to the centre of each spot
Results

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Chromatography can be used to separate and identify chloroplast pigments that have been
extracted from a leaf as each pigment will have a unique Rf value
The Rf value demonstrates how far a dissolved pigment travels through the stationary phase Your notes
Molecules with a higher affinity to the stationary phase, such as large molecules, will travel slower
and therefore have a smaller Rf value
Molecules that are more soluble in the mobile phase will travel faster and therefore have a larger Rf
value
Although specific Rf values depend on the solvent that is being used, in general:
Carotenoids have the highest Rf values (usually close to 1)
Chlorophyll b has a much lower Rf value
Chlorophyll a has an Rf value somewhere between those of carotenoids and chlorophyll b
Small Rf values indicate the pigment is less soluble and/or larger in size

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Your notes

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Your notes

Paper chromatography is used to separate photosynthetic pigments. These pigments can be identified
by their Rf values. In this example, a line of mixture (rather than a spot) is added to the paper.
Limitations
Paper chromatography is not as specific as other chromatography techniques
It is sufficient to separate and distinguish different pigments and to calculate their Rf value
Chromatography does not give data on the amount of each pigment present or the wavelengths that
they absorb
Colorimetry can be used to calculate these values

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Exam Tip
Your notes
Remember – the pigments themselves have colour (as described in the table). This is different from the
colours of light that they absorb.
Make sure you learn the approximate Rf values for the different pigments within chloroplasts (or at least
their values relative to each other). This means you should be able to identify different chloroplast
pigments based on their Rf values alone.

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5.6.5 The Light-Dependent Stage

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The Light-Dependent Stage
Photosynthesis occurs in two stages: the light-dependent stage, which takes place in the thylakoids,
and the light-independent stage, which takes place in the stroma
During the light-dependent stage of photosynthesis:
Light energy is used to breakdown water (photolysis) to produce hydrogen ions, electrons and
oxygen in the thylakoid lumen
A proton gradient is formed due to the photolysis of water resulting in a high concentration of
hydrogen ions in the thylakoid lumen
Electrons travel through an electron transport chain of proteins within the membrane
Reduced NADP (NADPH) is produced when hydrogen ions in the stroma and electrons from the
electron transport chain combine with the carrier molecule NADP
ATP is produced during a process known as photophosphorylation
Photophosphorylation & chemiosmosis
Photophosphorylation is the name for the overall process of using light energy and the electron
transport chain to phosphorylate ADP to ATP
The light-dependent reaction is sometimes called 'photophosphorylation'
During photophosphorylation, energetic (excited) electrons are passed along a chain of electron
carriers (known as the electron transport chain)
The electron carriers are alternately reduced (as they gain an electron) and then oxidised (as they lose
the electron by passing it to the next carrier)
The excited electrons gradually release their energy as they pass through the electron transport chain
The released energy is used to actively transport protons (H+ ions) across the thylakoid membrane,
from the stroma (the fluid within chloroplasts) to the thylakoid lumen (the space within thylakoids)
A ‘proton pump’ transports the protons across the thylakoid membrane, from the stroma to the
thylakoid lumen
The energy for this active transport comes from the excited electrons moving through the electron
transport chain
This creates a proton gradient, with a high concentration of protons in the thylakoid lumen and a low
concentration in the stroma
Protons then return to the stroma (moving down the proton concentration gradient) by facilitated
diffusion through transmembrane ATP synthase enzymes in a process known as chemiosmosis
This process provides the energy needed to synthesise ATP by adding an inorganic phosphate group
(Pi) to ADP (ADP + Pi → ATP)
The whole process is known as photophosphorylation as light provides the initial energy source for
ATP synthesis

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The photophosphorylation of ADP to ATP can be cyclic or non-cyclic, depending on the pattern of
electron flow in photosystem I or photosystem II or both
In cyclic photophosphorylation, only photosystem I is involved Your notes
In non-cyclic photophosphorylation, both photosystem I and photosystem II are involved
Photosystems are collections of photosynthetic pigments that absorb light energy and transfer the
energy onto electrons, each photosystem contains a primary pigment
Photosystem II has a primary pigment that absorbs light at a wavelength of 680nm and is
therefore called P680
Photosystem II is at the beginning of the electron transport chain and is where the photolysis of
water takes place
Photosystem I has a primary pigment that absorbs light at a wavelength of 700nm and is therefore
called P700
Photosystem I is in the middle of the electron transport chain
The energy carried by the ATP is then used during the light-independent reactions of
photosynthesis
Cyclic photophosphorylation
Cyclic photophosphorylation involves photosystem 1 (PSI) only
Light is absorbed by photosystem 1 (located in the thylakoid membrane) and passed to the
photosystem I primary pigment (P700)
An electron in the primary pigment molecule (ie. the chlorophyll molecule) is excited to a higher energy
level and is emitted from the chlorophyll molecule in a process known as photoactivation
This excited electron is captured by an electron acceptor, transported via a chain of electron carriers
known as an electron transport chain before being passed back to the chlorophyll molecule in
photosystem 1 (hence: cyclic)
As electrons pass through the electron transport chain they provide energy to transport protons (H+)
from the stroma to the thylakoid lumen via a proton pump
A build-up of protons in the thylakoid lumen can then be used to drive the synthesis of ATP from ADP
and an inorganic phosphate group (Pi) by the process of chemiosmosis

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Your notes

Cyclic photophosphorylation
Non-cyclic photophosphorylation
Light is absorbed by photosystem 2 (located in the thylakoid membrane) and passed to the
photosystem 2 primary pigment (P680)
Two electrons in the primary pigment molecule (ie. the chlorophyll molecule) are excited to a higher
energy level and are emitted from the chlorophyll molecule in a process known as photoionisation
Each excited electron is passed down a chain of electron carriers known as an electron transport
chain, before being passed on to photosystem 1
During this process chemiosmosis occurs:
The energy given by the electrons moving through the electron transport chain enables H­­+ ions
(protons) to pass from a low concentration in the stroma to a high
concentration in the thylakoid lumen
The creation of this proton gradient across the membrane later drives the synthesis of ATP in
photophosphorylation

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Photosystem 2 contains a water-splitting enzyme called the oxygen-evolving complex which

catalyses the breakdown (photolysis) of water by light:
Your notes
H2O → 2H+ + 2e- + ½O2
As the excited electrons leave the primary pigment of photosystem 2 and are passed on to
photosystem 1, they are replaced by electrons from the photolysis of water
At the same time as photoactivation of electrons in photosystem 2, electrons in photosystem 1 (PSI)
also undergo photoionisation
The excited electrons from photosystem 1 also pass along an electron transport chain, alternatively
reducing and oxidising proteins as they are accepted then passed on
These electrons combine with hydrogen ions (produced by the photolysis of water and transported
out of the thylakoid lumen by ATP synthase) and the carrier molecule NADP to give reduced NADP:
2H+ + 2e- + NADP → reduced NADP
The reduced NADP (NADPH) then passes to the light-independent reactions to be used in the
synthesis of carbohydrates
The electrons lost by photosystem 1 are replaced by the de-energised electrons from photosystem 2

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Your notes

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Your notes

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Your notes

Light-dependent photophosphorylation leads to the production of ATP and NADP

Comparison of Cyclic Photophosphorylation & Non-cyclic Photophosphorylation Table

Exam Tip
Make sure you know the difference between the two forms of photophosphorylation!Cyclic
photophosphorylation differs from non-cyclic photophosphorylation in two key ways:
Cyclic photophosphorylation only involves photosystem I (whereas non-cyclic
photophosphorylation involves photosystems I and II)
Cyclic photophosphorylation does not produce reduced NADP (whereas non-cyclic
photophosphorylation does)

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5.6.6 Using the Products of the Light-Dependent Reaction

Your notes
Using the Products of the Light-Dependent Reaction
Photosynthesis occurs in two stages: the light-dependent stage, which takes place in the thylakoids,
and the light-independent stage, which takes place in the stroma
During the light-dependent stage of photosynthesis:
Reduced NADP is produced when hydrogen ions combine with the carrier molecule NADP using
electrons from the photolysis of water
ATP is produced from ADP and Pi by ATP synthase in a process called photophosphorylation (ADP
+ Pi → ATP)
Photophosphorylation uses the proton (H+) gradient generated by the photolysis of water
Energy from ATP and hydrogen from reduced NADP are passed from the light-dependent stage
to the light-independent stage of photosynthesis
The energy and hydrogen are used during the light-independent reactions (known collectively as the
Calvin cycle) to produce complex organic molecules, including (but not limited to) carbohydrates,
such as:
Starch (for storage)
Sucrose (for translocation around the plant)
Cellulose (for making cell walls)

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Your notes

Photosynthesis occurs in two, closely-linked stages: the light-dependent stage, which takes place in
the thylakoids, and the light-independent stage, which takes place in the stroma

Exam Tip
Remember, the whole purpose of the light-dependent stage is to produce ATP and reduced NADP,
which are then used to complete the process of photosynthesis through the light-independent stage.

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5.6.7 The Light-Independent Stage

Your notes
The Light-Independent Stage
The light-independent stage of photosynthesis is sometimes referred to as the Calvin cycle
This stage produces complex organic molecules, including (but not limited to) carbohydrates, such as:
Starch (for storage)
Sucrose (for translocation around the plant)
Cellulose (for making cell walls)
The light-independent stage does not require energy from light and can therefore take place in light or
darkness. However, as it requires inputs of ATP and reduced NADP from the light-dependent stage, it
cannot continue indefinitely in darkness, as these inputs will run out
There are three main steps within 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 a reaction involving reduced NADP and ATP
RuBP is regenerated from TP in reactions that use ATP
Carbon fixation
Carbon dioxide combines with a five-carbon (5C) sugar known as ribulose bisphosphate (RuBP)
An enzyme called rubisco (ribulose bisphosphate carboxylase) catalyses this reaction
The resulting six-carbon (6C) compound is unstable and splits in two
This gives two molecules of a three-carbon (3C) compound known as glycerate 3-phosphate (GP)
The carbon dioxide has been ‘fixed’ (it has been removed from the external environment and has
become part of the plant cell)
Glycerate 3-phosphate (GP) is not a carbohydrate but the next step in the Calvin cycle converts it into
one
Reduction of glycerate 3-phosphate
Energy from ATP and hydrogen from reduced NADP – both produced during the light-dependent
stage of photosynthesis – are used to reduce glycerate 3-phosphate (GP) to a phosphorylated three-
carbon (3C) sugar known as triose phosphate (TP)
One-sixth of the triose phosphate (TP) molecules are used to produce useful organic molecules
needed by the plant
Regeneration of ribulose bisphosphate
Five-sixths of the triose phosphate (TP) molecules are used to regenerate ribulose bisphosphate
(RuBP)

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This process requires ATP

Your notes

The Calvin cycle

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Exam Tip
Your notes
Make sure to learn when in the Calvin cycle ADP and NADP are produced as you will be expected to
know it!

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5.6.8 Uses of Triose Phosphate

Your notes
Uses of Triose Phosphate
During the light-independent stage energy from ATP and hydrogen from reduced NADP (both
products of the light-dependent stage) are used to reduce glycerate 3-phosphate (GP) to a
phosphorylated three-carbon (3C) sugar known as triose phosphate (TP)
One-sixth of the triose phosphate (TP) molecules are used to produce useful organic molecules
needed by the plant:
Triose phosphates can condense to become hexose phosphates (6C), which can be used to
produce starch, sucrose or cellulose
Triose phosphates can be converted to glycerol while glycerate 3-phosphates can be converted
to fatty acids. These molecules join together to form lipids for cell membranes
Triose phosphates can be used in the production of amino acids for protein synthesis
Five-sixths of the triose phosphate (TP) molecules are used to regenerate ribulose bisphosphate
(RuBP). This process requires ATP

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5.6.9 Factors Affecting the Rate of Photosynthesis

Your notes
What are the Limiting Factors of Photosynthesis?
Plants need several factors for photosynthesis to occur:
The presence of photosynthetic pigments
A supply of carbon dioxide
A supply of water
Light energy
A suitable temperature
If there is a shortage of any of these factors, photosynthesis cannot occur at its maximum possible
rate
The main external factors that affect the rate of photosynthesis are:
Light intensity
Carbon dioxide concentration
Temperature
These are known as limiting factors of photosynthesis
If any one of these factors is below the optimum level for the plant, its rate of photosynthesis will be
reduced, even if the other two factors are at the optimum level
Although a lack of water can reduce the rate of photosynthesis, water shortages usually affect other
processes in the plant before affecting photosynthesis and is therefore not one of the main limiting
factors

Light intensity
When temperature and carbon dioxide concentration remain constant, changes in light intensity affect
the rate of photosynthesis
The rate of photosynthesis increases as light intensity increases:
The greater the light intensity, the more energy supplied to the plant and therefore the faster the
light-dependent stage of photosynthesis can occur
This produces more ATP and reduced NADP for the Calvin cycle (light-independent stage), which
can then also occur at a greater rate
During this stage of the graph below, light intensity is said to be a limiting factor of photosynthesis
At some point, if light intensity continues to increase, the relationship above will no longer apply and
the rate of photosynthesis will reach a plateau
At this point, light intensity is no longer a limiting factor of photosynthesis – another factor is limiting
the rate of photosynthesis
The factors which could be limiting the rate when the line on the graph is horizontal include
temperature being too low or too high, or not enough carbon dioxide

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Your notes

The effect of light intensity on the rate of photosynthesis

Carbon dioxide concentration
The rate of photosynthesis increases as carbon dioxide concentration increases:
Carbon dioxide is one of the raw materials required for photosynthesis
It is required for the light-independent stage of photosynthesis, when CO2 is combined with the
five-carbon compound ribulose bisphosphate (RuBP) during carbon fixation
This means the more carbon dioxide that is present, the faster this step of the Calvin cycle can
occur and the faster the overall rate of photosynthesis
This trend will continue until some other factor required for photosynthesis prevents the rate from
increasing further because it is in short supply
The natural level of CO2 in the atmosphere is 0.04%, it is therefore not advisable to increase the CO2
concentration much higher than this as it can become toxic
The factors which could be limiting the rate when the line on the graph is horizontal include
temperature being too low or too high, or not enough light

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Your notes

The effect of carbon dioxide concentration on the rate of photosynthesis

Temperature
As temperature increases the rate of photosynthesis increases, as the reaction is controlled by
enzymes
However, as the reaction is controlled by enzymes this trend only continues up to a certain
temperature, beyond which the enzymes begin to denature and the rate of reaction decreases
For most metabolic reactions, temperature has a large effect on reaction rate
For photosynthesis, temperature does not have a significant effect on the light-dependent
reactions, as these are driven by energy from light rather than the kinetic energy of the reacting
molecules
However, the Calvin cycle is affected by temperature, as the light-independent reactions are
enzyme-controlled reactions (e.g. rubisco catalyses the reaction between CO2 and the five-carbon
compound ribulose bisphosphate)
As long as there is enough light energy to produce ATP and NADPH in the light-dependent reaction,
increasing temperature up to an optimum temperature (this will vary by species and what its natural
habitat is) will increase the rate of the light-independent reactions and therefore the rate of
photosynthesis

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Although the rate of enzymatic reactions is the main component affected by temperature, other
components of the process can also be affected:
Increasing temperature causes stomata on the leaves to close in order to reduce water Your notes
loss; when the stomata are closed CO2 cannot enter the leaves and photosynthesis
will slow down
The light-dependent reaction relies on a proton gradient forming across the thylakoid
membrane; membrane permeability can be influenced by extreme temperatures, which may lead
to a dissipation of the proton gradient and a slowing down of photosynthesis

The effect of temperature on the rate of photosynthesis

The effect of the light intensity and carbon dioxide concentration on levels of GP,
TP and RuBP
The concentrations of glycerate 3-phosphate (GP), triose phosphate (TP) and ribulose bisphosphate
(RuBP) within chloroplasts can be affected by changes to light intensity and carbon dioxide
concentration

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A decrease in light intensity causes a decrease in TP and RuBP concentrations but a slight increase in
GP concentration
When there is less light available the light-dependent stage stops and does not form any more Your notes
products needed for the light-independent stage (ATP and NADPH)
As a consequence, GP builds up as it is not converted to TP
A lack of TP means that RuBP will not form
Over time the fixation of carbon dioxide will stop and the concentration of GP will plateau
Very low concentrations of carbon dioxide (less than 0.01%) causes a decrease in the concentration
of GP and TP but an increase in RuBP concentration
RuBP accepts carbon dioxide so when there is a lack of carbon dioxide molecules it remains
unfixed and builds up
The lack of carbon dioxide fixation prevents GP and TP molecules from forming

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Your notes

A decrease in light intensity and carbon dioxide concentration has different effects on the
concentrations of GP, TP and RuBP.
Agricultural practices balance limiting factors
An understanding of the effect of limiting factors on the rate of photosynthesis can be used to
increase crop yields in protected environments, such as glasshouses
In the most sophisticated glasshouses, for example, sensors can be used to monitor the light intensity,
the humidity of the atmosphere and the carbon dioxide concentration around the crops
This means that plants could continue to grow through the night if they are kept lit with artificial
lighting
Plants can be grown out of their natural season and habitat because the temperature can be kept
constant all year round
All these factors can be managed by a computer and their levels adjusted to ensure the crop can
photosynthesis at the highest rate possible
Water can be supplied by irrigation systems throughout the glasshouse or fields which sometimes
contain added fertilisers or growth nutrients such as nitrates to aid plant growth
Natural pests that may spread disease or eat the crops can be controlled within agricultural settings by
pesticides or by separating the plants from the unfiltered outside air
This maximises the yield of the crop
Farmers have to find a balance between crop yield and the cost of maintaining 24-hour lighting and
year-round heating as well as the environmental implications this has

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Exam Tip
Your notes
Interpreting graphs of limiting factors can be confusing for many students, but it’s quite simple.In the
section of the graph where the rate is increasing (the line is going up), the limiting factor is whatever the
label on the x-axis (the bottom axis) of the graph is.In the section of the graph where the rate is not
increasing (the line is horizontal), the limiting factor will be something other than what is on the x-axis –
choose from temperature, light intensity or carbon dioxide concentration.

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5.6.10 Practical: Investigating Factors Affecting the Rate of

Photosynthesis Your notes

Practical: Investigating Factors Affecting the Rate of Photosynthesis

Investigations to determine the effects of light intensity, carbon dioxide concentration and
temperature on the rate of photosynthesis can be carried out using aquatic plants, such as Elodea or
Cabomba (types of pondweed)
The effect of these limiting factors on the rate of photosynthesis can be investigated in the following
ways:
Light intensity – change the distance (d) of a light source from the plant (light intensity is
proportional to 1/d2)
Carbon dioxide concentration – add different quantities of sodium hydrogencarbonate
(NaHCO3) to the water surrounding the plant, this dissolves to produce CO2
Temperature (of the solution surrounding the plant) – place the boiling tube containing the
submerged plant in water baths of different temperatures
Whilst changing one of these factors during the investigation (as described below), ensure the other
two remain constant
For example, when investigating the effect of light intensity on the rate of photosynthesis, a glass
tank should be placed in between the lamp and the boiling tube containing the pondweed to
absorb heat from the lamp – this prevents the solution surrounding the plant from changing
temperature
Apparatus
Distilled water
Test tube
Beaker
Lamp
Aquatic plant, algae or algal beads
Ruler
Sodium hydrogen carbonate solution
Thermometer
Test tube plug
Syringe
Method
Ensure the water is well aerated before use by bubbling air through it
This will ensure oxygen gas given off by the plant during the investigation form bubbles and do not
dissolve in the water

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Ensure the plant has been well illuminated before use

This will ensure that the plant contains all the enzymes required for photosynthesis and that any
changes of rate are due to the independent variable Your notes
Set up the apparatus in a darkened room
Ensure the pondweed is submerged in sodium hydrogen carbonate solution (1%) – this ensures
the pondweed has a controlled supply of carbon dioxide (a reactant in photosynthesis)
Cut the stem of the pondweed cleanly just before placing into the boiling tube
Measure the volume of gas collected in the gas-syringe in a set period of time (eg. 5 minutes)
Change the independent variable (ie. change the light intensity, carbon dioxide concentration or
temperature depending on which limiting factor you are investigating) and repeat step 5
Record the results in a table and plot a graph of volume of oxygen produced per minute against the
distance from the lamp (if investigating light intensity), carbon dioxide concentration, or temperature

The effect of light intensity on an aquatic plant is measured by the volume of oxygen produced
Results - Light Intensity

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The closer the lamp, the higher the light intensity (intensity ∝ 1/d2)
Therefore, the volume of oxygen produced should increase as the light intensity is increased
At a point, the volume of oxygen produced will stop changing even if the light is moved closer Your notes
This is when the light stops being the limiting factor and the temperature or concentration of
carbon dioxide is limiting the rate of photosynthesis
The effect of these variables could then be measured by increasing the temperature of water (by
using a water bath) or increasing the concentration of sodium hydrogen carbonate respectively
The results should be displayed on a graph of light intensity vs. rate of photosynthesis
Rate of photosynthesis = volume of oxygen produced ÷ time elapsed
Limitations
Algae is often used in experiments on photosynthesis and respiration rates but it can be very hard to
maintain consistency in the number of algae and it can be hard to handle directly in the water
Immobilised algae beads are beads of jelly with a known surface area and volume that contain
algae, therefore it is easier to ensure a standard quantity
Immobilised algae beads are easy and cheap to grow, they are also easy to keep alive for several
weeks and can be reused in different experiments
The method is the same for algae beads though it is important to ensure sufficient light coverage
for all beads
Practical: Measuring the rate of the light-dependent stage of photosynthesis
The light-dependent reactions of photosynthesis take place in the thylakoid membrane and involve
the release of high-energy electrons from chlorophyll a molecules
These electrons are picked up by the electron acceptor NADP in a reaction catalysed by the enzyme
dehydrogenase
However, if a redox indicator (such as DCPIP or methylene blue) is present, the indicator takes up the
electrons instead of NADP
This causes the indicator to change colour
DCPIP: oxidised (blue) → accepts electrons → reduced (colourless)
Methylene blue: oxidised (blue) → accepts electrons → reduced (colourless)
The colour of the reduced solution may appear green because chlorophyll produces a green
colour
The rate at which the redox indicator changes colour from its oxidised (blue) state to its reduced
(colourless) state can be used as a measure of the rate of dehydrogenase activity and therefore, the
rate of the light-dependent stage of photosynthesis
When light is at a higher intensity, or at more preferable light wavelengths, the rate of
photoactivation of electrons is faster, therefore the rate of reduction of the indicator is faster

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Your notes

The light activates electrons from chlorophyll molecules during the light-dependent reaction. Redox
indicators accept the excited electrons from the photosystem, becoming reduced and therefore
changing colour.
Apparatus
Leaves
Isolation medium
Pestel and mortar
Lamp
Test tubes
Stopwatch

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Aluminium Foil
DCPIP or methylene blue indicator
Buffer solution Your notes
Method – Measuring light as a limiting factor
Leaves are crushed in a liquid known as an isolation medium
This produces a concentrated leaf extract that contains a suspension of intact and functional
chloroplasts
The medium must have the same water potential as the leaf cells so the chloroplasts don’t shrivel
or burst and contain a buffer to keep the pH constant
The medium should also be ice-cold (to avoid damaging the chloroplasts and to maintain
membrane structure)
The experiment should be set up in a dark room so that the light source and intensity can be controlled
The room should be at an adequate temperate for photosynthesis and maintained throughout, as
should carbon dioxide concentration
Small tubes are set up with different intensities, or different colours (wavelengths) of light shining on
them
If different intensities of light are used, they must all be of the same wavelength (same colour of
light) – light intensity is altered by changing the distance between the lamp and the test tube
If different wavelengths of light are used, they must all be of the same light intensity – the lamp
should be the same distance in all experiments
DCPIP or methylene blue indicator is added to each tube, as well as a small volume of the leaf extract
A control that is not exposed to light (wrapped in aluminium foil) should also be set up to ensure the
affect on colour is due to the light
The time taken for the redox indicator to go colourless (or green, as the chlorophyll may also colour the
solution) is recorded
This is a measure of the rate of photosynthesis
Results
A graph should be plotted of absorbance against time for each distance from the light
As the light intensity decreases, the rate of photosynthesis also decreases
This is because the lowered light intensity will slow the rate of photoionisation of the chlorophyll
pigment, so the overall rate of the light dependent reaction will be slower
This means that less electrons are released by the chlorophyll, hence the DCPIP accepts less
electrons. This means that it will take longer to turn from blue to colourless
When the DCPIP is blue, the absorbance is higher. The rate at which the absorbance decreases can
therefore be used to determine the activity of the dehydrogenase enzyme

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A higher rate of decrease, shown by a steep gradient on the graph, indicates that the
dehydrogenase is highly active.
Your notes
Limitations
This experiment is not measuring the rate of dehydrogenase activity directly (through measuring the
rate of substrate use or product made) but is instead predicting what the rate would be by measuring
the rate of electron transfer from the photosystems
The concentration of DCPIP will depend on the number of chloroplasts in a sample and therefore the
number of light-dependent electron transport chains
It is therefore important to control the amount of leaf used to produce the chloroplast sample and
also how much time is spent crushing the leaf to release the chloroplast
It is also a good idea to measure a specific wavelength absorption by each sample on the
colorimeter before and after the experiment so you can get a more accurate change in oxidised
DCPIP concentration
Results should also be repeated and the mean value calculated
The time taken to go colourless is subjective to each person observing and therefore one person
should be assigned the task of deciding when this is

Exam Tip
Learn the 3 limiting factors and how each one can be altered in a laboratory environment:
Light intensity – the distance of the light source from the plant (intensity ∝ 1/d2)
Temperature - changing the temperature of the water bath the test tube sits in
Carbon dioxide - the amount of NaHCO3 dissolved in the water the pondweed is in
Also, remember that the variables not being tested (the control variables) must be kept constant.

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