40

3. Pigment Regeneration - electron replacement

from the splitting of H2O in oxygenic photosynthesis.

The 2 main stages are light-dependent reactions and light-independent reactions (Calvin
cycle).
 41

#83 Chloroplasts
Photosynthesis takes place inside chloroplasts.

These are organelles surrounded by 2 membranes, called an envelope.

Chloroplasts are found in mesophyll cells in leaves:

- Palisade mesophyll cells contain most chloroplasts.

- Spongy mesophyll cells and Guard cells also contain chloroplasts.

 42

Lamellae and light-dependent reactions

The membranes inside a

chloroplast are called lamellae,
and it is here that the light-
dependent reactions take place.
The membranes contain
chlorophyl molecules, arranged in
groups called photosystems.
There are two kinds of
photosysterns, PSI and PSII, each
of which contains slightly different
kinds of chlorophyll.

There are enclosed spaces between pairs of membranes, forming fluid-filled sacs
called thylakoids. These are involved in photophosphorylation - the formation of ATP
using energy from light. Thylakoids are often arranged in stacks called grana (singular:
granum).

Stroma and light-independent reactions

The 'background material' of the chloroplast is

called the stroma, and this is where the light-
independent reactions take place.

Chloroplasts often contain starch

grains and lipid droplets. These are stores of
energy-containing substances that have been
made in the chloroplast but are not immediately
needed by the cell or by other parts of the plant.
 43

#84 Photosynthetic Pigments

Photosynthetic pigments are pigments presented in

chloroplasts or photosynthetic bacteria. They capture light
energy necessary for photosynthesis and convert it
to chemical energy.

Pigments
 A pigment is any substance that absorbs light.
 The color of the pigment comes from the wavelengths of light that are reflected
(not absorbed).
 If pigments absorb all wavelengths they will appear black.
 If pigments reflect most of the wavelengths they will appear white.
 The light absorption pattern of a pigment is called the absorption spectrum.
When pigments absorb light, electrons are temporarily boosted to a higher energy level.
Energized electrons move further from the nucleus of the atom. When the e- returns to a
lower energy level the energy may be:
 dissipated as heat
 re-emitted as a longer wavelength of light - fluorescence
 captured in a chemical bond (carbon gain!)

Photosynthetic pigments in chloroplast

Choloroplats contain several different pigments,

which absorb different wavelengths of light. The
photosynthetic pigments of higher plants form 2
groups: the cholophylls and the caroteinoids.

1. Chlorophylls absorb mainly red and blue-

violet light, reflect green light - giving green
leaves their colour.
 44

2. Carotenoids: orange pigments that

protect chlorophyll from damage by the
formation of single oxygen atoms (free
radicals). They can also absorb
wavelengths of light that chlorophyll cannot
absorb, and pass on some of the energy
from the light to chlorophyll. They absorb
strongly in the blue-violet range.
Carotenoids are usually masked by the
green chlorophylls.

There are 2 types of carotenoid:

 carotenes (β-carotene)
 xantophylls.

Main and accessory photosynthetic pigments

- Main pigment

 Chlorophyll a is the most abundant pigment in most plants. Its absorption peaks
are 430nm (blue) and 662nm (red). It emits an electron when it absorbs light.

- Accessory pigments

 Chlorophyll b is similar to chlorophyll a, but its absorption peaks are 453nm and
642nm. It has a similar role to chlorophyll a, but is not as abundant.
 Carotenoids : carotene and Xanthophylls.
The combination of all of the pigments increases the range of colors that plants can use
in photosynthesis.
 45

Chlorophill molecule.

Absorption spectrum and action spectrum of chloroplast pigments

 46

An absorption spectrum is a graph showing the percentage of light absorbed by

pigments, for each wavelength of light.

An example is the absorption spectrum of chlorophyll a and b.

 The best absorption is seen with violet-blue light.

 There is also good absorption with red-orange light.
 Most of the green-yellow light is reflected and therefore not absorbed. This
wavelength of light shows the least absorption.

The action spectrum of photosynthesis is a graph showing the rate of

photosynthesis for each wavelength of light. The rate of photosynthesis will not be the
same for every wavelength of light.

 The rate of photosynthesis is the least with green-yellow light (525 nm-625 nm).
 Red-orange light (625nm-700nm) shows a good rate of photosynthesis.
 The best rate of photosynthesis is seen with violet-blue light (400nm-525nm).

The wavelengths that is does not absorb are reflected from it.
Chlorophyll Is the main pigment contained in chloroplasts. It looks green because it
reflects green light. Other wavelengths (colours) of light are absorbed.
llle diagram shows the wavelengths of light absorbed by the various pigments found
in chloroplasts. These graphs are called absorption spectra.
If we shine light of various wavelengths on chloroplasts containing different pigments,
we can measure the rate at which they give off oxygen. These graphs are called action
spectra.
 47

# 85 The light-dependent reactions, Photophosphorilation

Chlorophyll molecules in photosystern I (PSI) and photosystern II (PSII) absorb light

energy. The energy excites electrons, raising their energy level so that they leave the
chlorophyll. The chlorophyll is said to be photo-activated.

PSII contains an enzyme that splits water when activated by light. This reaction is
called photolysis ('splitting by light'). The water molecules are split into oxygen and
hydrogen atoms. Each hydrogen atom then loses its electron, to become a positively
charged hydrogen ion (proton), H+.

The electrons are picked up by the chlorophyll in PSII, to replace the electrons they lost.
The oxygen atoms join together to form oxygen molecules, which diffuse out of the
chloroplast and into the air around the leaf.
 48

The light- dependent reactions. Credit: Pears education.

The electrons emitted from PSII are picked up by electron carriers in the membranes of
the thylakoids. They are passed along a chain of these carriers, losing energy as they
go. The energy they lose is used to make ADP combine with a phosphate group,
producing ATP. This is called photophosphorylation. At the end of the electron carrier
chain, the electron is picked up by PSI, to replace the electron the chlorophyll in PSI had
lost.

The electrons from PSI are passed along a different chain of carriers to NADP. The
NADP also picks up the hydrogen ions from the split water molecules. The NADP
becomes reduced NADP.

We can show all of this in a diagram called the Z-scheme. The higher up the diagram,
the higher the energy level. If you follow one electron from a water molecule, you can
see how it
• is taken up by PSII
• has its energy raised as the chlorophyll in PSII absorbs light energy
• loses some of this energy as it passes along the electron carrier chain
• is taken up by PSI
• has its energy raised agaln as the chlorophyll in PSI absorbs light energy
• becomes part of a reduced NADP molecule

At the end of this process, two new substances have been made. These are ATP and
reduced NADP. Both of them will now be used in the next stage of photosynthesis, the
light-independent reactions.
 49

Non-cyclic and cyclic photophosphorylation

The sequence of events just described and shown in the flow diagram above is known
as non-cyclic photophosphorylation.

There is an alternative pathway for the electron that is emitted from PSI. It can simply be
passed along the electron transport chain, then back to PSI again. ATP is produced as it
moves along the electron transport chain (photophosphorylation). However, no reduced
NADP is produced. This is called cyclic photophosphorylation.
 50

Z- cheme.

Video: Photosynthesis
https://www.youtube.com/watch?v=YeD9idmcX0w
 51

#86 The light-independent reactions (Calvin cycle)

The light-independent reactions take place in the stroma

of the chloroplast, where the enzyme ribulose
bisphosphate carboxylase, usually known
as rubisco, is found.

1. Carbon fixation

CO2 diffuses into the stroma from the air spaces within
the leaf. It enters the active site of rubisco, which
combines it with a 5-carbon compound called ribulose
bisphosphate, RuBP. The reaction is
called carbon fixation.

The products of this reaction are two 3-carbon molecules,

glycerate 3-phosphate, GP.
 52

2. Reduction

Energy from ATP and hydrogen from reduced NADP are then used to convert the GP
into triose phosphate, TP. Triose phosphate is the first carbohydrate produced in
photosynthesis.

3. RuBP regeneration

Most of the triose phosphate is used to produce ribulose bisphosphate (RuBP), so that
more carbon dioxide can be fixed.

The rest is used to make glucose or whatever other organic substances the plant cell
requires. These include:
 polysaccharides such as starch for energy storage and cellulose for making cell
walls,
 sucrose for transport,
 amino acids for making proteins,
 lipids for energy storage
 nucleotides for making DNA and RNA.
 53

#87 Separating chlorophyll pigments by Thin layer chromatography

(TLC)

Chromatography is a method of separation that relies

on the different solubilities of different solutes in a
solvent. A mixture of chlorophyll pigments is
dissolved in a solvent, and then a small spot is placed
onto chromatography paper. The solvent gradually
moves up the paper, carrying the solutes with it. The
more soluble the solvent, the further up the paper it is
carried.

Paper chromatography is a useful technique in the separation and identification of

different plant pigments.
 In this technique, the mixture containing the pigments to be separated is first
applied as a spot or a line to the paper about 1.5 cm from the bottom edge of the paper.
 The paper is then placed in a container with the tip of the paper touching the
solvent. Solvent is absorbed by the paper and moves up the paper by capillary action.
 As the solvent crosses the area containing plant pigment extract, the pigments
dissolve in and move with the solvent.
 The solvent carries the dissolved pigments as it moves up the paper. The
pigments are carried along at different rates because they are not equally soluble.
Therefore, the less soluble pigments will move slower up the paper than the more
soluble pigments. This is known as developing a chromatogram.

There are various methods. The one described here uses Thin layer
chromatography (TLC) on specially prepared strips instead of paper. It is a
chromatography technique for analyzing mixtures by separating the compounds in the
mixture. TLC can be used to help determine the number of components in a mixture, the
identity of compounds, and the purity of a compound. During chromatography, a mobile
phase (eluent) distributes the compounds present in a mixture over a stationary phase
(adsorbent).

The distance traveled by a particular compound can be used to identify the compound.
The ratio of the distance traveled by a compound to that of the solvent front is known as
the Rf value; unknown compounds may be identified by comparing their Rf's to the Rf's
of known standards.
 54

Rf equation:

Only an outline of the procedure is given here, so you cannot use these instructions to
actually carry out the experiment.
 55

Video TLC
https://www.youtube.com/watch?v=CmHFVxTxkGs

https://www.youtube.com/watch?v=rbp_Qc4jMAc
 56

#88 Limiting factors in photosynthesis

A limiting factor is a factor that controls a

process. Light intensity,
temperature and, CO2 concentration and
availability of H2O are all factors which can
control the rate of photosynthesis.

Usually, only one of these factors will be the limiting factor in a plant at a certain time.
This is the factor which is the furthest from its optimum level at a particular point in time.
If we change the limiting factor the rate of photosynthesis will change but changes to the
other factors will have no effect on the rate.

If the levels of the limiting factor increase so that this factor is no longer the furthest
from its optimum level, the limiting factor will change to the factor which is at that point in
time, the furthest from its optimum level. For example, at night the limiting factor is likely
to be the light intensity as this will be the furthest from its optimum level. During the day,
the limiting factor is likely to switch to the temperature or the carbon dioxide
concentration as the light intensity increases.
 57

Effects of changes in light intensity, CO2, H2O and temperature on the rate of
photosynthesis

1. Light intensity

 This affects the rate of the light-

dependent reaction. The energy that drives
this process is light energy.
 When the light intensity is poor, there is
a shortage of ATP and NADPH, as these are
products from the light dependent reactions.
Without these products the light independent
reactions can't occur as glycerate 3-phosphate
cannot be reduced. Therefore a shortage of
these products will limit the rate of
photosynthesis.

2. Temperature

 This affects the rate of the light-

independent reaction. The energy that drives
this process is heat energy.
 At higher temperatures, molecules have
more kinetic energy so collide more often and
are more likely to react when they do collide.
 Many enzymes are involved during the
process of photosynthesis. At low temperatures
these enzymes work slower. At high
temperatures the enzymes no longer work
effectively. This affects the rate of the reactions
in the Calvin cycle and therefore the rate of
photosynthesis will be affected.

3. CO2 concentration

 CO2 is a reactant in photosynthesis.

Normal air contains only about 0.04% CO2.
 When the CO2 concentration is low, the
amount of glycerate 3-phosphate produced is
limited as CO2 is needed for its production and
therefore the rate of photosynthesis is affected.
 58

4. Availability of H2O

H2O is a reactant in photosynthesis, but there is usually far more H2O available than
CO2, so even if water supplies are low this is not usually a problem. However, water
supply can affect the rate of photosynthesis indirectly, because a plant that is short of
water will close its stomata, preventing CO2 from diffusing into the leaf.

lf the level of anyone of these factors is too low, then the rate of photosynthesis will be
reduced. The factor that has the greatest effect in reducing the rate is said to be the
limiting factor.

Economics of greenhouses

Farmers can use their knowledge of factors limiting the rate of photosynthesis
to increase crop yields. This is particularly true in greenhouses, where the conditions
are more easily controlled than in the open air outside:

 The use of artificial light allows photosynthesis to continue beyond daylight

hours. Bright lights also provide a higher-than-normal light intensity.
 The use of artificial heating allows photosynthesis to continue at an increased
rate.
 The use of additional CO2 released into the atmosphere inside the greenhouse
also allows photosynthesis to continue at an increased rate.

Artificial light in the green house.

However, the additional cost of providing extra lighting, heat and CO2 has to be weighed
against the increased crop yield and the extra income it will provide. The cost of should
not exceed the additional income it generates for the farmer.
 59

In practice, the farmer will need to find the optimum growing conditions for the crop,
given the costs of providing extra lighting, heat and CO2. Paraffin lamps have
traditionally been used in greenhouses. Their use increases the rate of photosynthesis
because as well as the light generated from the lamps, the burning paraffin produces
heat and CO2 too.

Investigating the effect of environmental factors on the rate of photosynthesis

One way to measure the rate of photosynthesis is to measure the rate at which oxygen
is given off by an aquatic plant. There are various ways in which oxygen can be
collected and measured. One method is shown in the diagram below.

Alternatively, you can make calcium alginate balls containing green algae and place
them in hydrogencarbonate indicator solution. As the algae photosynthesise, they take
in carbon dioxide which causes the pH around them to increase. The indicator changes
from orange, through red to magenta.

Whichever technique is used, you should change one factor (your independent variable)
while keeping all others constant (the control variables). The dependent variable will be
the rate at which oxygen is given off (measured by the volume of oxygen collected per
minute in the capillary tube) or
the rate at which carbon dioxide is used (measured by the rate of change of colour of
 60

the hydrogencarbonate indicator solution).

The independent variables you could investigate are:

 Light intensity. You can vary this by using a lamp to shine light onto the plant or
algae. The closer the lamp. the higher the light intensity.
 Wavelength of light. You can vary this by placing coloured filters between the
light source and the plant. Each filter will allow only light of certain wavelengths to pass
through.
 CO2 concentration. You can vary this by adcting sodium hydrogencarbonate to
the water around the aquatic plant. This contains hydrogencarbonate Ions, which are
used as a source of carbon dioxide by aquatic plants.
 Temperature. The part of the apparatus containing the plant or algae can be
placed in a water bath at a range of controlled temperatures.

Video: Limiting factors of photosynthesis

https://www.youtube.com/watch?v=go8V2GQq268
 61

#89 Summary of Photosynthesis

1 In photosynthesis, light energy is absorbed by
chlorophyll pigments and converted to chemical
energy, which is used to produce complex organic
molecules.

2 In the light-dependent reactions, water is split by

photolysis to give hydrogen ions, electrons and
oxygen. The hydrogen ions and electrons are used to
reduce the carrier molecule, NADP, and the oxygen is
given off as a waste product.

3 ATP is synthesised in the light-dependent reactions

of cyclic and non-cyclic photophosphorylation. During
these reactions the photosynthetic pigments of the
chloroplast absorb light energy and give out excited
electrons. Energy from the electrons is used to
synthesise ATP.

4 ATP and reduced NADP are the two main products of the light-dependent reactions of
photosynthesis, and they then pass to the light-independent reactions.

5 In the light-independent reactions, carbon dioxide is trapped by combination with a 5C

compound, RuBP, which acts as an acceptor molecule. This reaction is catalysed by the
enzyme ribulose bisphosphate carboxylase (rubisco), which is the most common
enzyme in the world. The resulting 6C compound splits to give two molecules of a 3C
compound, GP (also known as PGA). GP is reduced to carbohydrate, using ATP and
reduced NADP from the light-dependent reactions. This carbohydrate can be converted
into other carbohydrates, amino acids and lipids or used to regenerate RuBP. This
sequence of light-independent events is called the Calvin cycle.

6 Chloroplasts, palisade mesophyll cells and whole leaves are all adapted for the
efficient absorption of light for the process of photosynthesis.

7 When a process is affected by more than one factor, the rate of the process will be
limited by the factor closest to its lowest value. The rate of photosynthesis is subject to
various such limiting factors, including light intensity and wavelength, carbon dioxide
concentration and temperature.

8 A graph of the particular wavelengths of light that are absorbed by a photosynthetic

pigment is called an absorption spectrum, and a graph of the rate of photosynthesis at
different wavelengths of light is called an action spectrum.

9 The different pigments present in a chloroplast can be separated by paper

chromatography.