Photosynthesis: Overview

 Photosynthesis is a series of chemical reactions that occurs

in producers such as plants and algae
o Producers are also known as autotrophs; organisms that make their
own organic compounds
 Photosynthesis converts light energy into chemical energy which is then
stored in the biomass of producers
 The light energy is used to split strong bonds in water
molecules (H2O), releasing hydrogen and oxygen
 Oxygen is released into the atmosphere as a waste product
 Hydrogen is combined with carbon dioxide to produce glucose
o Chemical energy is stored within the bonds in glucose molecules;
glucose can therefore function as a fuel for respiration
o It can be said that hydrogen is stored in glucose molecules
 ATP as an Energy Carrier in Photosynthesis
 All organisms require a constant supply of energy to maintain their cells and
stay alive
 This energy is required e.g.
o For building new molecules from the products of digestion
during anabolic reactions
o To move substances across cell membranes in active transport or to
move substances within cells
o For muscle contraction
o In the conduction of nerve impulses
 In all known forms of life the molecule adenosine triphosphate, or ATP, is
used to transfer and supply energy within cells
o ATP is therefore known as the universal energy currency
o ATP diffuses within cells to where it is needed

 ATP is a type of nucleic acid and is structurally very similar to the nucleotides
that make up DNA and RNA
o It is a phosphorylated nucleotide
 A nucleotide consists of a nitrogenous base, a sugar, and
a single phosphate group
 ATP contains three phosphate groups, hence triphosphate

ATP contains adenine, a ribose sugar, and three phosphates molecules.

Removal of one phosphate creates ADP, and removal of two phosphates
creates AMP.
  ATP is produced by the addition of inorganic phosphate (Pi), a type of
phosphate group, to adenosine diphosphate, or ADP

ADP + Pi ATP


o ADP contains two phosphate groups, hence diphosphate
 ATP can be produced when the passage of electrons along a series of
proteins known as the electron transport chain releases energy for
the phosphorylation of ADP
o This process occurs in the mitochondria during respiration and in
chloroplasts during photosynthesis
 In photosynthesis the energy originally gained by the electrons
in this process comes from light, so this method of ATP
production is known as photophosphorylation
 Photo = light

 The hydrolysis, or breakdown, of ATP releases an inorganic phosphate as

well as a small amount of energy which can be used by the cell

ATP ADP + Pi


o The removal of a phosphate group is known as dephosphorylation
o The hydrolysis of ATP is catalysed by the enzyme ATPase
 The ADP and inorganic phosphate produced by the hydrolysis of ATP can
be recycled to make more ATP

ADP + Pi ATP

ATP is formed during respiration and can be hydrolysed to release energy for
processes such as active transport, muscle contraction, and building new
molecules (anabolic reactions). ATP can then be regenerated from ADP and
phosphate.
 Photosynthesis: Light-Dependent Stage
 Photosynthesis takes place in two distinct stages
o The light-dependent reactions, which rely on light directly
o The light-independent reactions, which do not use light directly, though do rely on
the products of the light-dependent reactions
 Both these sets of reactions take place within the chloroplast
o The light-dependent reactions take place across the thylakoid membrane
o The light-independent reactions take place in the stroma
 Light energy in the light-dependent reactions enables the splitting of water molecules in a
reaction known as photolysis
o Photolysis of one molecule of water, or H2O, produces
 2 hydrogen ions (2H+), also known as protons
 2 electrons (2e-)
 One atom of oxygen (O)
o The hydrogen ions and electrons are used during the light-dependent reactions while
the oxygen is given off as a waste product
 During the light-dependent reactions light energy is converted into chemical energy in the
form of ATP and reduced NADP
o NADP is a type of molecule called a coenzyme; its role is to transfer hydrogen from
one molecule to another
o When NADP gains hydrogen it is reduced, and can be known as either reduced
NADP or NADPH
o Remember that
 Reduction is gain of electrons, gain of hydrogen, or loss of oxygen
 Oxidation is loss of electrons, loss of hydrogen, or gain of oxygen
o Reduced NADP can reduce other molecules by giving away hydrogen
o NADP can oxidise other molecules by receiving hydrogen
 The useful products of the light-dependent reactions, ATP and NADPH, are transferred
to the light-independent reactions within the chloroplast

The products of the light-dependent reaction are ATP, NADPH, and oxygen.
Oxygen is given off as a waste product while ATP and NADPH pass to the
light-independent reactions. The ADP and NADP produced during the light-
independent reaction can pass back to the light-dependent reactions to allow
more ATP and NADPH to be produced.

Production of ATP and NADPH

 ATP and NADPH are produced during the light-dependent reactions as a result of a series of
events that occur on the thylakoid membrane known as photophosphorylation
o Photo = light
o Phosphorylation = the addition of phosphate; in this case to ADP to form ATP
 Two types of photophosphorylation take place
o Non-cyclic photophosphorylation
 This produces both ATP and NADPH
o Cyclic photophosphorylation
 This produces ATP only
 Both cyclic and non-cyclic photophosphorylation involve
o A series of membrane proteins which together make up the electron transport
chain
 Electrons pass from one protein to another along the electron transport chain,
releasing energy as they do so
o Chemiosmosis
 The energy released as electrons pass down the electron transport chain is
used to produce ATP

Non-cyclic photophosphorylation

 Light energy hits photosystem II in the thylakoid membrane

o It is slightly confusing that photosystem II comes first in this sequence; the numbers
simply reflect the order in which the photosystems were discovered
 Two electrons gain energy and are said to be excited to a higher energy level
 The excited electrons leave the photosystem and pass to the first protein in the electron
transport chain
o As the excited electrons leave photosystem II they are replaced by electrons from
the photolysis of water
 The electrons pass down the chain of electron carriers known as an electron transport
chain
 Energy is released as the electrons pass down the electron transport chain which
enables chemiosmosis to occur
o H+ ions are actively pumped from a low concentration in the stroma to a high
concentration in the thylakoid space, generating a concentration gradient across
the thylakoid membrane
o H+ ions diffuse back across the thylakoid membrane into the stroma via ATP
synthase enzymes embedded in the membrane
o The movement of H+ ions causes the ATP synthase enzyme to catalyse
the production of ATP
 At the end of the electron transport chain the electrons from photosystem II are passed to
photosystem I
 Light energy also hits photosystem I, exciting another pair of electrons which leave the
photosystem
 The excited electrons from photosystem I also pass along an electron transport chain
 These electrons combine with hydrogen ions from the photolysis of water and
the coenzyme NADP to form reduced NADP

H+ + 2e- + NADP+ → NADPH

 The reduced NADP and the ATP pass to the light-independent reactions
 Non-cyclic photophosphorylation involves photosystems I and II and
produces both ATP and NADPH

Cyclic photophosphorylation

 Light hits photosystem I

 Electrons are excited to a higher energy level and leave the photosystem
 The excited electrons pass along the electron transport chain, releasing energy as they do
so
 The energy released as the electrons pass down the electron transport chain provides energy
to drive the process of chemiosmosis
o H+ ions are actively pumped from a low concentration in the stroma to a high
concentration in the thylakoid space, generating a concentration gradient across the
thylakoid membrane
o H+ ions diffuse back across the thylakoid membrane into the stroma via ATP
synthase enzymes embedded in the membrane
o The movement of H+ ions cause the ATP synthase enzyme to catalyse
the production of ATP
 At the end of the electron transport chain the electrons rejoin photosystem I in a complete
cycle; hence the term cyclic photophosphorylation
 The ATP produced enters the light-independent reaction

Cyclic photophosphorylation involves Photosystem I and produces ATP

 Fixation of Carbon from Carbon Dioxide
 The light-independent reactions of photosynthesis are sometimes referred to as
the Calvin cycle
 The reactions eventually allow for the production of complex organic molecules such as
o Starch for storage
o Sucrose for transport
o Cellulose for making cell walls
 The light-independent reactions do not require energy from light but do
require ATP and reduced NADP from the light-dependent reactions
 There are three main steps within the light-independent reactions

1. Carbon dioxide is combined with ribulose bisphosphate (RuBP), a 5-carbon (5C)

compound; this yields two molecules of glycerate 3-phosphate (GP), a 3-carbon
(3C) compound
2. GP is reduced to glyceraldehyde 3-phosphate (GALP), another 3C compound, in
a reaction involving reduced NADP and ATP
3. RuBP is regenerated from GALP in reactions that use ATP

Carbon dioxide and RuBP are combined

 Carbon dioxide combines with a 5C sugar known as RuBP in a reaction catalysed by the
enzyme rubisco
 The resulting 6-carbon (6C) compound is unstable and splits in two
 This results in two molecules of a 3C compound known as glycerate 3-phosphate (GP)
 The carbon dioxide has been ‘fixed’, meaning that it has been removed from the external
environment and become part of a molecule inside the plant cell

Reduction of glycerate 3-phosphate

 Energy from ATP and hydrogen from reduced NADP, both produced during the light-
dependent reactions, are used to reduce the two 3C molecules of GP to two 3C
molecules known as GALP
 Some of the carbons in GALP go towards the production of useful organic molecules
such as glucose, while the rest remain in the Calvin cycle to allow the regeneration of
RuBP
o Two molecules of GALP contain six carbon atoms, five of which are needed to
regenerate RuBP; this means that for every turn through the Calvin cycle only one
sixth of a molecule of glucose is produced
o Glucose is a 6-carbon molecule, so six turns of the Calvin cycle are required to
produce one molecule of glucose

Regeneration of ribulose bisphosphate

 Five sixths of the GALP molecules are used to regenerate RuBP

 This process requires ATP
The Calvin cycle produces glucose and other important biological molecules

Products of the Light-Dependent Stage

 Intermediate molecules of the Calvin cycle, such as glyceraldehyde 3-phosphate
(GALP), are used to produce various other biological molecules needed by plants, such
as:
 o Hexose sugars e.g. glucose
 Glucose can enter the respiration reactions during which ATP is produced
 Hexose sugars can be converted into other hexose sugars e.g. glucose can
be converted to sucrose for transport in the phloem
 Hexose sugars can be joined to make polysaccharides such
as starch and cellulose
o Glycerol can be used for building lipid molecules such as triglycerides and
phospholipids
o Fatty acids which form the tails of lipid molecules such as triglycerides and
phospholipids
o Nucleic acids form the basis of DNA and RNA
 Phosphates from the soil are combined with the molecules of the Calvin
cycle to produce nucleic acids
o Acetyl coenzyme A is important coenzyme in respiration
o Amino acids which can be used in protein synthesis for building polypeptides
 Nitrates from the soil need to be combined with the molecules of the
Calvin cycle for amino acids to be produced
 Many of the molecules produced are used to build new plant biomass; these molecules
are passed on to consumers when plant tissue is eaten

The products of photosynthesis include amino acids, polysaccarides, lipids and

nucleic acids

Chloroplasts: Structure & Function

 Chloroplasts are the organelles in plant cells where photosynthesis occurs
 Each chloroplast is surrounded by a double-membrane known as the chloroplast envelope
o Each of the envelope membranes is a phospholipid bilayer
 Chloroplasts are filled with a cytoplasm-like fluid known as the stroma
o The stroma contains enzymes and sugars, as well as ribosomes and chloroplast
DNA
o If the chloroplast has been photosynthesising there may be starch grains or lipid
droplets in the stroma
 A separate system of membranes is found in the stroma
 o This membrane system consists of a series of flattened fluid-filled sacs known
as thylakoids, each surrounded by a thylakoid membrane
o Thylakoids stack up to form structures known as grana (singular granum)
o Grana are connected by membranous channels called lamellae (singular lamella),
which ensure the stacks of sacs are connected but distanced from each other
 Several components that are essential for photosynthesis are embedded in the thylakoid
membranes, including:
o ATP synthase enzymes
o Proteins called photosystems that contain photosynthetic pigments such as
chlorophyll a, chlorophyll b, and carotene

Chloroplasts are the site of photosynthesis

Chloroplast structure is related to function

 Chloroplast envelope
o The double membrane encloses the chloroplast, keeping all of the components
needed for photosynthesis close to each other
o The transport proteins present in the inner membrane control the flow of molecules
between the stroma and cytoplasm
 Stroma
o The gel-like fluid contains enzymes that catalyse the reactions of photosynthesis
 DNA
o The chloroplast DNA contains genes that code for some of the proteins used in
photosynthesis
 Ribosomes
o Ribosomes enable the translation of proteins coded by the chloroplast DNA
 Thylakoid membrane
o There is a space between the two thylakoid membranes known as the thylakoid
space, in which conditions can differ from the stroma e.g. a proton gradient can be
established between the thylakoid space and the stroma
o The space has a very small volume so a proton gradient can develop very quickly
 Grana
o The grana create a large surface area, maximising the number of photosystems and
allowing maximum light absorption
 o Grana also provide more membrane area for proteins such as electron carriers and
ATP synthase enzymes, which together enable the production of ATP
 Photosystems
o There are two types of photosystems; photosystem I and photosystem II,
containing different combinations of photosynthetic pigments such as chlorophyll
a, chlorophyll b, and carotene
o Each photosystem absorbs light of a different wavelength, maximising light
absorption e.g. photosystem I absorbs light at a wavelength of 700 nm while
photosystem II absorbs light at a wavelength of 680 nm

Absorption & Action Spectra

 Chloroplasts contain several different photosynthetic
pigments within photosystems embedded in the thylakoid membranes
 Different pigments absorb light of different wavelengths
o Chlorophylls absorb wavelengths in the blue-violet and red
regions of the light spectrum, reflecting green light and appearing
green in colour
o Carotenoids absorb wavelengths of light mainly in the blue-violet
region of the spectrum, reflecting yellow and orange light
 Carotenoids often remain in leaves after the breakdown of
chlorophyll in the autumn, giving some leaves their yellow,
orange, and red autumn colours

Examples of Photosynthetic pigments Table

 The amount of light at different wavelengths absorbed by a particular

pigment gives that pigment's absorption spectrum (plural spectra)
o Because each type of pigment absorbs light at different wavelengths
the absorption spectrum of each pigment is different
Different photosynthetic pigments absorb light of different wavelengths,
giving different absorption spectra

 A plant's rate of photosynthesis varies depending on the wavelengths of

light available
 The changing rate of photosynthesis at different wavelengths is known as
an action spectrum
 Action spectra are very closely correlated to the absorption spectra of the
different pigments
o Having several different pigments with different absorption spectra
allows plants to photosynthesise under a wider variety of light
wavelengths; this extends the action spectra of plants and
maximises rates of photosynthesis
Plant action spectra are closely related to the absorption spectra of the
different photosynthetic pigments

Separation of Photosynthetic Pigments with

Chromatography
 Chloroplasts contain several different photosynthetic
pigments within photosystems embedded in their thylakoid membranes
 Photosynthetic pigments absorb different wavelengths of light, so are different in colour
o The colour of a pigment is due to the wavelengths of light reflected by that pigment,
e.g. chlorophylls absorb light at the red and blue ends of the visible spectrum and
reflect light in the green part of the spectrum, so appear as green pigments
 Chromatography can be used to separate and identify chloroplast pigments that have
been extracted from a leaf

Chromatography

 Chromatography is a technique that is used to separate mixtures

 o Different components within a mixture travel through materials at different speeds
due to their size or charge
o This causes different components to separate
o An Rf value can be calculated for each component of the mixture on the basis of its
rate of movement
 Two of the most common techniques for separating photosynthetic pigments are
o Paper chromatography
 The mixture of pigments is passed through paper made of cellulose
o Thin-layer chromatography (TLC)
 The mixture of pigments is passed through a thin layer of an adsorbent, e.g.
silica gel
 The pigments travel faster than through paper, so they separate more
distinctly

Apparatus

 Leaf sample
 Dropping pipette
 Acetone
 Pestle and mortar
 Filter paper or TLC paper
 Pencil
 Ruler
 Capillary tube
 Beaker or boiling tube
 Chromatography solvent

Method

1. Draw a straight line in pencil approximately 1cm above the bottom of the paper being used,
and use the pencil to draw a dot in the middle of the line; this marks where you will place the
leaf sample
o Do not use a pen as the ink will separate into pigments within the experiment and
obscure the results
2. Cut a section of leaf and place it in a mortar
o It is important to choose a healthy leaf that has been in direct sunlight so you can be
sure it contains many active photosystems
3. Add 20 drops of acetone and use the pestle to grind up the leaf sample and release the
pigments
o Acetone is an organic solvent and therefore fats, such as the phospholipid
membranes in plant cells, dissolve in it
o Acetone and mechanical pressure are used to break down the cell, chloroplast and
thylakoid membranes to release the pigments
4. Extract some of the pigment using a capillary tube and spot it onto the dot in the centre of the
pencil line you have drawn
5. Suspend the paper over a beaker containing a small amount of chromatography solvent; the
end of the paper closest to the pigment extract needs to touch the chromatography solvent,
but the level of the solvent should be below the pencil line at this stage
o The solvent will move up the paper
o The pigment mixture will be dissolved in the solvent and carried with the solvent as
it moves
6. Leave the paper suspended in the solvent until the solvent has almost reached the top of the
paper
7. Remove the paper from the solvent and draw a pencil line marking the level of the solvent on
the paper
 o The solvent may continue moving after the paper is removed from it, so it is important
to draw a pencil line immediately
o The pigments 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
8. Calculate the Rf value for each pigment spot

Rf value = distance travelled by pigment ÷ distance travelled by the solvent


o Always measure to the centre of each spot of pigment

Results

 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 is a measure of how far a dissolved pigment travel
o Larger, less soluble molecules will travel more slowly and therefore have a smaller
Rf value
o Smaller, more soluble molecules will travel faster and therefore have a larger
Rf value
 Although specific Rf values depend on the solvent that is being used, in general
o Carotenoids have the highest Rf values, usually close to 1
o Chlorophyll b has a much lower Rf value
o Chlorophyll a has an Rf value somewhere between those of carotenoids and
chlorophyll b
Chromatography can be used to separate photosynthetic pigments, which can
then be identified by their Rf values.