Photosynthesis

Chloroplast Ultrastructure

Chloroplasts are small flattened organelles, they absorb light to carry out photosynthesis which produces sugar the plant can use for respiration to provide energy. They have a double membrane, the outer membrane is permeable to small molecules (water, carbon dioxide, oxygen) and ions, which diffuse easily but is not permeable to larger proteins. The inner membrane regulates the passage of large substances (sugars and proteins) in and out of the chloroplast through membrane bound transport proteins.

The inside of a chloroplast is filled with a gel-like fluid called stroma, it contains enzymes, starch granules, proteins and chloroplast DNA and ribosomes. Starch granules are used to store the products of photosynthesis. 

The stroma contains disc shaped fluid filled sacs made of thylakoid membrane called thylakoids. The inside of the sacs is hollow and known as the thylakoid space, it contains the chlorophyll and other pigments required for absorbing light for photosynthesis as well as the enzymes required for the light dependent reactions. The thylakoids are stacked like coins into structures called grana (sing. granum). The grana are linked by bits of thylakoid membrane called lamellae (sing. Lamella). The thylakoid membranes contain the ATP synthase enzymes required to make ATP in the light dependent reactions. 

Where do the different stages of photosynthesis occur?

The light dependent stage occurs in the thylakoid membrane of the chloroplast, and the light independent stage occurs in the stroma of the chloroplast. 

Photosynthetic Pigments

Photosynthetic pigments are arranged in structures called photosystems. Photosystems are protein complexes found in the thylakoid membranes.

Photosystems contain coloured photosynthetic pigments which absorb light energy needed for photosynthesis, there is a chlorophyll a molecule at the centre of each photosystem, chlorophyll absorbs red wavelengths of light. Photosystem I (PSI) contains a chlorophyll which absorbs light with a wavelength of 700nm best, photosystem II (PSII) contains a chlorophyll which absorbs light with a wavelength of 680nm best. 

Plants have several photosynthetic pigments in their leaves because each pigment absorbs different wavelengths of light. Having more pigments increases the range of wavelengths that plants can absorb so it increases their ability to photosynthesise. In the photosystems these accessory pigments are not directly involved in the light dependent reactions but they channel more captured light energy to chlorophyll so more electrons can be excited.

Some plants produce high levels of anthocyanins, dark red and purple pigments which can help protect the plant from high UV radiation. These pigments are also used to colour flowers and fruits e.g blueberries. Different species of plants will have different proportions of photosynthetic pigments in their leaves. 

Chromatography

Chromatography is a method used to separate molecules in mixtures. The molecules are dissolved and move through the mobile phase (a liquid solvent). Different molecules will spend different amounts of time in the mobile or stationary phase. The longer the components spend in the mobile phase the faster or further up the plate they move, this allows them to be separated out. The rates of migration of individual pigments will depend on three things: 

1. Solubility - Pigments (solutes) dissolve in the solvent

  - The more soluble the pigment is in the solvent, the further it will travel

2. Affinity to stationary phase -  Pigment molecules interact with the plate 

 - Molecules which interact more strongly with the plate will not travel as far

3. Mass -  Pigment molecules are also separated by size

 - The smaller the molecule the further it will travel

R_f value

The distance the substance moved through the stationary phase relative to the distance the solvent moved can be calculated to give an R_f value. Each photosynthetic pigment will have a specific R_f value when run under certain conditions. Pigments that travel further up the paper will have a higher Rf value. R_f values can be compared to known R_f values to identify the pigment.

Why might multiple chromatograms need to be carried out?

However, more than one substance can have the same Rf value for a particular solvent and chromatography paper and so it’s possible that multiple chromatograms will need to be run with different solvents (and/or chromatography paper) in order to find out the exact identity of the substance. 

How does chromatography work?

Solvents will compete with compounds for sites on the stationary phase, chromatography works because the stationary phase will always be more polar than the mobile phase. This is why it is important to use a non-polar solvent where possible because a less polar solvent will not compete well, allowing the compounds to remain bound to the stationary phase. A polar solvent will compete well with molecules and will occupy sites on the stationary phase. This will force compounds into the mobile phase, and result in an increased travel distance.

Chromatography Method

1. Tear up a sample of plant leaves into small pieces, place in a mortar with a pinch of sand, and grind with a pestle.  

2. Add solvent and continue to grind with a pestle. It is important that the tissue be ground up to break open plant cells and release the chloroplasts.

3. Gently draw a pencil line 1cm from the base of the chromatography paper. The pencil line marks the starting position of the pigments. It is important it is in pencil so that the line doesn’t move with the solvent and won’t mask the results of the chromatography.

4. Repeatedly spot a small quantity of the leaf extract onto the centre of the line, allowing the spot to dry between each application. The spot is made with small quantities through repeated spotting and drying in order to build up a concentrated, but still small, spot.   

5. Suspend the chromatography plate/paper from a bung in the glass vial with 1cm depth of solvent in the bottom. Ensure that the chromatography plate dips into the solvent but the spot of leaf extract remains above the surface of the solvent. The spot needs to be above the surface of the solvent so that the pigments travel up the plate and don’t just dissolve out into the solvent in the vial.

6. Place a lid on the container. Chemicals used for solvents are very volatile and the vapours can be hazardous. 

7. Allow the solvent to run up the chromatography plate until the solvent front is near the bung then remove the plate, mark the location of the solvent front with a pencil, and allow to dry. The solvent evaporates quickly and so the extent to which it has travelled up the plate needs to be marked whilst it can be seen otherwise the distance will be unknown and the R_F value cannot be calculated. Drying ensures that the movement of solvent and pigments stops.

8. The resultant chromatography plate is called a chromatogram which can then be photographed or the location of each separated pigment can be marked with pencil. The pigments can be affected by light and fade so the measurements need to be taken quickly and the spots marked so they can be identified once the spot fades. 

What is a photosynthetic pigment?

Coloured molecules that absorb the light energy needed for photosynthesis.

Where are photosynthetic pigments found?

In the thylakoid membrane

How are photosynthetic pigments arranged?

Into photosystems in the thylakoid membrane

What is the main photosynthetic pigment?

Chlorophyll a

Why do photosystems contain accessory pigments?

To help absorb the wavelengths of light that are not easily absorbed by chlorophyll  (maximise light absorption)

Why does chlorophyll appear green?

It absorbs light at two wavelengths 430nm (blue) and 662nm (red). It reflects green light strongly so it appears green.

Why is it important to use an organic (non-polar) solvent and not a polar one like water?

A polar solvent could compete with the dissolved molecules for space on the stationary phase and could cause them to move further than they should do, giving the incorrect R_f value.

Why is it important to mark positions in pencil rather than pen (particularly the starting position of the concentrated extract)?

Ink may dissolve in the solvent move up the stationary phase. Colours could affect the results and there will be no visible start line to measure R_F value from.

Why is it important that the spot of plant extract is above the surface of the solvent when the chromatography plate is placed in the vial?

To ensure the pigments are carried up by the solvent and don’t just dissolve out into the solvent at the bottom.

Why is repeated application of the plant extract onto the same spot required?

To increase the concentration of the pigments

Why is it important that the chromatogram is stopped before the solvent front reaches the top?

So that the distance travelled by the solvent (solvent front) can be identified and drawn – needed for the R_f value calculation

Why is it important to mark the solvent front quickly?

The solvent is volatile and will evaporate quickly, once dry it can’t be seen.

Why is it useful to mark the positions of the pigment spots or take a photograph of the chromatogram soon after it has been run?

Light can cause them to fade

Photosynthesis is a metabolic pathway (it takes place in a series of enzyme-controlled reactions) it can be split into two main parts that take part in two different places in the chloroplast.

The light-dependent reactions take place in the thylakoid membrane, they require light energy absorbed by chlorophyll to excite electrons which raises their energy level and that energy is released to produce a small amount of ATP from ADP (photophosphorylation). 

The light-independent reactions take place in the stroma and it uses the products of the light-dependent reactions along with CO₂ to make glucose. 

 Light Dependent Reactions: Photolysis

When photosystem II is left positively charged – the electrons must be replaced. They are provided from the photolysis of water. The energy from light is used to split water into protons (H⁺ ions), electrons and oxygen. Only PSII can split water as it has the correct enzyme complex. 

• Light hits water and causes it to split producing H+, e- and O2

• H+ from water is accepted by NADP to make red. NADP

• The electrons from photolysis of water replace the lost electrons from the chlorophyll

• The O2 from photolysis of water is released out of the stomata or is used in respiration

Light Dependent Reaction:

Photoionisation describes the process of using light energy to excite electrons in chlorophyll enough so that they can leave the molecule. It leaves the chlorophyll as a positively charged ion. Photosystem II:

• Light energy is absorbed by chorophyll in PSII and excites the electrons in the molecule

• The electrons rise up energy levels

• Some of the high energy electrons escape from the chlorophyll molecule

• The high-energy electrons which leave the chlorophyll are taken up by an electron carrier

• The high-energy electrons are passed down the electron transport chain in the thylakoid membranes

• As the electrons move down the transport chain, energy is released

• The released energy is used to actively transport H+ into the thylakoids creating a concentration gradient across the membrane

• The H+ diffuses down the concentration gradient out through the thylakoid membranes

• Energy from the diffusion allows the ATP synthase enzyme associated with the thylakoid membranes to make ATP from ADP+ Pi. This is the chemiosmotic theory

Photosystem I:

• PSI absorbs light energy and excites the electrons again to an even higher level

• These excited electrons are transferred with a H+ from the photolysis of water to reduce NADP

• ATP and red. NADP are used in the light independent stage

Light Dependent Reactions: The Electron Transport Chain

Redox reactions reminder! – OILRIG 

• oxidation is loss of electrons or protons OR gained oxygen

• reduction is gain of electrons or protons OR lost oxygen

• redox reactions occur together, oxidation of one molecule always involves reduction of another. 

Co-enzymes

Hydrogen or electrons are transported by the coenzyme NADP, which transfers them from one molecule to another. NADP becomes NADPH or reduced NADP when it collects a hydrogen and it can then reduce other molecules. 

Phosphorylation and ATP

Adenosine triphosphate (ATP) is soluble and easily diffuses around the cell to provide energy for cellular processes e.g active transport, DNA replication, cell division, protein synthesis photosynthesis. It can be broken down and remade with a simple reaction. 

ATP is synthesised using an enzyme called ATP synthase which catalyses the condensation reaction between ADP and an inorganic phosphate. Adding a phosphate molecule is phosphorylation. In this case, light is used as an energy source, so the process is called photophosphorylation.

The process of photophosphorylation

ATP is produced by adding an inorganic phosphate to ADP. In photosynthesis energy from excited electrons and H⁺ ions generated from photolysis are used to phosphorylate ADP. 

The two photosystems are linked by electron carriers, these are proteins which can transfer electrons between them using a series of redox reactions. The chain of photosystems and proteins that electrons flow through in the thylakoid membranes are known as an electron transport chain. 

Light Independent Reactions: Calvin Cycle

The light-independent reaction is the second stage of photosynthesis, the light independent reactions are also known as the Calvin cycle and take place in the stroma of chloroplasts. It can happen without light, but it does require the products of the light dependent reaction. The Calvin cycle uses carbon dioxide to produce two molecules of triose phosphate (TP) which can be used to make glucose and other organic substances.

The light independent reaction:

• The key molecule in the light independent stage is RuBP ( a 5C molecule)

• CO2 is accepted by RuBP to make an unstable 6C molecule. This requires the use of an enzyme rubisco.

• The unstable 6C molecule breaks down to form 2 molecules of GP (2×3C molecules)

• GP is reduced to make triose phosphate- GP reduced to triose phosphate requires red NADP and ATP from the light-dependent stage. ATP and the H⁺ ions (provided by reduced NADP) from the light-dependent reaction are needed to do this. The products of the light-dependent stage, red NADP and ATP diffuse into the stroma.

• There are two fates of the triose phosphate- Triose phosphate can be converted into organic sugars like glucose (6C). Two TP molecules are needed to make one hexose sugar (6C). Triose phosphate can be used to regenerate RuBP so the Calvin Cycle can carry on indefinitely. The rest of the ATP produced in the light-dependent reaction is used to regenerate RuBP.

Products of the Calvin cycle: 

1. NADP (passes back into the light-dependent reaction)

2. ADP (passes back into the light-dependent reaction)

3. Inorganic Phosphate (passes back into the light-dependent reaction)

4. TP (used to build useful organic compounds such as glucose, amino acids and lipids)

5. RuBP is regenerated and reused in the Calvin cycle

Efficiency of the system:

One hexose sugar (e.g glucose) is made by joining 2 molecules of TP. Three turns of the Calvin cycle produces 6 molecules of TP, but 5 of these are needed to regenerate RuBP. This means that three turns actually only produces one molecule of TP that can be used to make glucose so the Calvin cycle actually needs to happen 6 times to make one hexose sugar.

Therefore, for every three turns of the cycle only one molecule of TP is produced.

Limiting Factors of Photosynthesis

Optimum growing conditions for photosynthesis:

1. Light Intensity and Wavelength

The higher the light intensity the more energy it provides to excite electrons, however at extremely high light intensities e.g. high UV chlorophyll may also be damaged. Photosynthetic pigments in plants mostly only absorb red and blue light from sunlight (green light is reflected) this can be low in the shade/low light. 

2. Temperature

Photosynthesis is a series of reactions that rely on enzymes (ATP synthase and Rubisco). If the temperature is too low (below 10°C) the enzymes will work too slowly, too high (above 45°C) the enzymes may start to denature. Remember: denaturing causes bonds holding the protein chains in an enzyme to break, thus changing its’ tertiary structure so that it is no longer a complimentary shape to the substrate. No enzyme-substrate complexes can occur. 

These temperatures can be different for different plant species, as some plants are adapted to live in conditions that are outside this range e.g desert. At higher temperatures stomata also close to avoid water loss which decreases CO₂ availability which slows down photosynthesis. 

3. Carbon dioxide levels

Atmospheric CO₂ concentrations are low (0.04%), photosynthesis in plants works best when this level is increased to 0.4%. If this is increased further however, stomata will begin to close. 

4. Availability of water

Plants need a constant supply of water but not too much as their roots can become waterlogged. This can cause the roots to “drown” as they are not able to respire underwater so no ATP can be made for active transport of minerals. This can affect the plants health and often leads to chlorosis (yellowing of the leaves) because not enough magnesium is being absorbed to make chlorophyll. This can reduce photosynthesis

How Limiting Factors Affect the Rate of Photosynthesis: Light Intensity

From A-B light intensity is the limiting factor (e.g night to daylight) as it increases so does the rate they are directly proportional. 

Point B represents the saturation point – increasing the light intensity will not make a difference to the rate, something else has become the limiting factor.

Temperature

Both graphs level off when light intensity is no longer the limiting factor but the graph at 25°C levels off at a higher point. The lower temperature must have been a limiting factor as the rate was not as fast at 15°C. 

CO₂ Concentration

As with temperature above, both graphs level off when the light intensity is no longer the limiting factor. The temperature was the same for both so the difference in the graph heights must have been caused by the CO₂ concentration. The graph at 0.4% CO₂ levels off at a higher point so CO₂ concentration must have been the limiting factor in the bottom graph where the rate was not as fast. 

Farmers try to optimise conditions to improve growth:

Growers can create optimum conditions in glasshouses to increase the rate of photosynthesis and improve yield of their crops. Higher levels of photosynthesis = more sugars/starch = more energy = more growth/fruit formation:

• They increase carbon dioxide concentration in the air using CO₂ generators

• They increase the light intensity during the winter/cloudy days using lamps, they can use LEDs to only give the plants blue/red wavelengths of light. 

• The glasshouses trap heat energy from the sun which warms the air but to keep the temperature optimum heating or cooling systems such as fans, automatic windows, and air circulation systems can be used. 

In order to make sure that any changes to a limiting factor are having an effect all three factors must be measured and the two not being changed must be controlled to make sure they are not affecting the results. Growers will also want to consider cost of these technologies vs the profit they will make with them.