Photosynthesis

It is a physiological process used by plants, algae and some bacteria to convert light energy into chemical energy.

Organisms use this chemical energy to synthesise large organic molecules, which forms the building blocks of living cells, from simple inorganic molecules such as water and carbon dioxide- this is autotrophic nutrition.

Organisms that photosynthesise are called 'photoautotrophs', because they use light as the energy source for autotrophic nutrition. These organisms are also described as 'producers' because they are at the first trophic level of a food chain and provide energy and organic molecules to other, non-photosynthetic organisms.

A photon is a particle of light; each photom contains an amount (a quantum) of energy. The  main product of photosynthesis is monosccharide sugar, which can be converted to disaccharides for transport and then to starch for storage. 

Photosynthesis is an example of carbon fixation (carbon --> sugars). The carbon for synthesising all types of organic molecule is provided by this. It is an endothermic process, so requires energy. It also needs electrons, which makes it a reduction reaction. It helps to regulate the concentration of CO2 in the atmosphere and oceans.

Plants and other organisms that photosynthesise also respire. During respiration, they oxidise the organic molecules that they have previously synthesised by photosynthesis and stored, releasing chemical energy.

Non-photosynthetic organisms such as fungi, animals, many protoctists and many types of bacteria are described as 'heterotrophs'. They obtain energy by digesting complex organic molecules of food to smaller molecules than they can use as respiratory substrates. They obtain energy from the products of digestion by respiration.

During respiration, glucose and other organic compounds are oxidised to produce carbon dioxide and water. Respiration releases chemical energy (it is exothermic) that can drive the organism's metabolism:

C6H12O6 + 6O2 ------> 6H2O + 6H2O + energy

Both photosynthesis and aerobic respiration are important in cycling carbon dioxide and oxygen in the atmosphere.

The products of one process are the raw materials for the other process: aerobic respiration removed oxygen from the atmosphere and adds carbon dioxide, while photosynthesis does the opposite.

Plants respire all the time, however they only synthesise during day light.

Plants often compete with each other for light.

The intensity of light has to be sufficient to allow photosynthesis at a rate that replenishes the carbohydrate stores used up by respiration.

When photosynthesis and respiration proceed at the same rate, so that there is no net gain or loss of carbohydrate, the plant is at its compensation point.

The time a plant takes to reach its compensation point is called the compensation period. This period is different for different plant species.

Shade plants utilise light of lower intensity than sun plants can.

When exposed to light after being in darkness, shade plants reach their compensation point sooner (shorter compensation period) than sun plants, which require a higher light intensity to achieve their optimum rate of photosynthesis.

Most chloroplasts are disk shaped and around 2-10um long.

Each is surrounded by a double membrane, the envelope, with an intermembrane space of width 10-20nm between the inner and outer membrane.

The outer membrane is highly permeable.

There are 2 distinct regions, visible on electron micrographs, inside a chloroplast: the fluid-filled matrix called the stroma and the grana that consists of stacks of thylakoid membranes.

The first stage of photosynthesis, the light dependent stage, takes place in the grana.

Chloroplasts have 3 distinct membranes- outer, inner and thylakoid, giving 3 separate internal compartments- the intermembrane space, stroma and thylakoid space.

The thylakoids within a granum may be connected to thylakoids within another granum by intergranal lamellae. The thylakoid membrane of each chloroplast is less permeable and is folded into flattened disc-like sacs called thylakoids that form stacks. Each stack of thylakoids is called a granum. One granum may contain up to 100 thylakoids. With many grana in every chloroplast and with many chloroplasts in each photosynthetic cell, there is a huge surface area for:

• the distribution of the photosystems that contain the photosynthetic pigments that trap sunlight energy.
• the electron carriers and ATP synthase enzymes needed to convert that light energy into ATP.

Proteins embedded in the thylakoid membranes hold the photosystems in place. 

The grana are surrounded by the stroma, so the products of the light dependent stage can easily pass to the stroma to be used in the light independent stage.

The stroma is the fluid filled matrix.

It contains the enzymes needed to catalyse the reactions of the light-independent stage of photosynthesis, as well as starch grains, oil droplets, small ribosomes and DNA.

The loop of DNA contains genes that code for some of the proteins needed for photosynthesis.

These proteins are assembled at the chloroplast ribosomes.

Within the thylakoid membranes of each chloroplast are funnel-shaped structures called photosystems.

These photosystems contain photosynthetic pigments.

Each pigment absorbs light of a particular wavelength and reflects other wavelengths of light.

Each pigment appears, to our eyes and brain, the colour of the wavelength of light it is reflecting.

The energy associated within the wavelengths of light captured is funnelled down to the primary pigment reaction centre, consisting of a type of chlorophyll, at the base of the photosystem. 

Chlorophylls are a mixture of pigments.

All have a similar molecular structure, consisting of a porphyrin group, in which is a magnesium atom and a long hydrocarbon chain.

There are 2 forms of chlorophyll a- both of which appear blue-green.

Both are situated at the centre of photosystems.

Both absorb red light, but they have different absorption peaks:

• P680 is found in photosystem II and its peak of absorption is light of wavelength 680nm.
• P700 is found in photosystem I and its peak of absorption is light of wavelength 700nm.

Chlorophyll a also absorbs some blue light, of wave length around 440nm.

Chlorophyll b absorbs light of wavelengths 400-500nm and around 640nm. It appears yellow green. 

Carotenoids absorb blue light of wavelengths 400-500nm.

They reflect yellow and orange light.

Xanthophylls absorb blue and green light of wavelengths 375-550nm.

They reflect yellow light.

The photosynthetic pigments in plants can be separated using thin layer chromatography (TLC). It involves a mobile phase (a liquid solvent) and a stationary phase (the chromatography plate). 

1. Grind up several leaves with some anhydrous sodium sulphate and some propanone.

2. Transfer the liquid to a test tube, add some petroleum ether and shake the tube. 2 distict layers will form in the liquid- the top layer is the pigments mixed in with the petroleum ether.

3. Transfer some of the liquid from the top layer into a second test tube with some anhydrous sodium sulphate.

4. Draw a horizontal pencil line near the bottom of a chromatography place. Build up a single spot of the liquid from step 3 on the line by applying several drops and ensuring each one is dry before adding the next one. This is the point of origin.

5. Once the point of origin is completely dry, put the plate into a glass beaker with some prepared solvent (e.g. a mixture of propanone, cyclohexane and petroleum ether)- just enough so that the point of origin is a little above the solvent. Put a lid on the beaker and leave the plate to develop. As the solvent spreads up the plate, the different pigments move with it, but at different rates- so they separate.

6. When the solvent has nearly reached the top, take the plate out and mark the solvent front (furthest point where the solvent reached) with a pencil and leave the plate to dry in a well-ventilated place.

7. There should be several new coloured spots on the chromatography plate between the point of origin and the solvent front. These are the separated pigments. You can calculate their Rf values and look them up in a database to identify what the pigments are.

Rf value = Distance travelled by spot / Distance travelled by solvent

Occurs in the grana (thylakoids) of chloroplasts and involves the photosystems. It involves the direct use of light energy.

This stage consists of:

1. Light harvesting at the photosystems

2. Photolysis of water

3. Photophosphorylation- the production of ATP in the presence of light

4. the formation of reduced NADP.

Oxygen, the by-product of photosynthesis, is also produced in the light-dependent stage.

• In photosystem I (PSI), the pigment at the primary reaction centre is a type of chlorophyll a, which has a peak absorption of red light of wavelength 700nm.

• In photosystem II (PSII), the pigment at the primary reaction centre is also a type of chlorophyll a, but this has a peak absorption of red light of wavelength 680nm.

In PSII, there is an enzyme that, in the presence of light, splits water molecules into protons, electrons and oxygen.

It is called photolysis: 2H20 --> 4H+ + 4e- + O2.

Some of the oxygen produced during photolysis is used by plant cells for aerobic respiration, but during periods of high light intensity the rate of photosynthesis is greater than the rate of respiration in the plant, so much of the oxygen by-product will diffuse out of the leaves, through stomata, into the surrounding atmosphere.

Water:

•is the source of protons (H+ ions) that will be used in photophosphorylation

•donates electrons to chlorophyll to replace those lost when light strikes chlorophyll

•is the source of the by-product, oxygen

•keeps plant cells turgid, enabling them to function

Photophosphorylation is the generation of ATP from ADP and inorganic phosphate, in the presence of light.

There are 2 types of photophosphorylation:

• Non-cyclic photophosphorylation involves PSI and PSII. It produces ATP, oxygen and reduced NADP.
• Cyclic photophosphorylation involves PSI only. It produces ATP but in smaller quantities than in non-cyclic photophosphorylation.

Both involve iron-containing proteins embedded in the thylakoid membranes that accept and donate electrons and form an electron transport system.

1. When a photon of light strikes PSII, its energy is channelled to the primary pigment reaction centre.

2. The light energy excites a pair of electrons inside the chlorophyll molecule.

3. The energised electrons escape from the chlorophyll molecule and are captured by an electron carrier, which is a protein with iron at its centre, embedded in the thylakoid membrane.

4. These electrons are replaced by electrons derived from photolysis.

5. When this iron ion combines with an electron it becomes reduced (Fe2+). It can then donate the electron, becoming reoxidised (Fe3+), to the next electron carrier in the chain.

6. As electrons are passed along a chain of electron carriers embedded in the thylakoid membrane, some energy associated with the electrons is released at each step.

7. This energy is used to pump protons across the thylakoid membrane into the thylakoid space.

8. Eventually the electrons are captured by another molecule of chlorophyll a in PSI. These electrons replace those lost from PSI due to excitation by light energy.

9. A protein-iron-sulfur complex called ferredoxin accepts the electrons from PSI and passes  them to NADP in the stroma.

10. As protons accumulate in the thylakoid space, a protein gradient forms across the membrane.

11. Protons diffuse down their concentration gradient through special channels in the membrane associated with ATP synthase enzymes and, as they do so, the flow of protons causes ADP and inorganic phosphate to join, forming ATP.

12. As the protons pass through the channel they are accepted, along with electrons, by NADP which become reduced. The reduction of NADP is catalysed by the enzyme NADP reductase.

The light energy has been converted into chemical energy in the form of ATP by photophosphorylation. ATP and reduced NADP are now in the stroma ready for the light independent stage of photosynthesis.

This only uses PSI.

As light strikes PSI, a pair of electrons in the chlorophyll molecule at the reaction centre gain energy and become excited.

They escape from the chlorophyll and pass to an electron carrier system and then pass back to PSI.

During the passage of electrons along the electron carriers, a small amount of ATP is generated.

However, no photolysis of water occurs, so no protons or oxygen are produced.

No reduced NADP is generated.

Chloroplasts in guard cells contain only PSI. They produce only ATP which actively brings potassium ions into the cells, lowering the water potential so that water follows by osmosis. This causes the guard cells to swell and opens the stoma.

The light independent stage of photosynthesis takes place in the stroma of chloroplasts.

Although is does not directly use light energy, it uses the products of the light-dependent stage.

If the plant is not illuminated, the light-independent stage soon ceases, because ATP and hydrogen are not available to reduce the carbon dioxide and synthesise large complex organic molecules.

Carbon dioxide is the source of carbon for the production of all organic molecules found in all the carbon-based life forms on Earth.

These organic molecules may be used as structures (e.g. cell membranes, antigens, enzymes, muscle proteins, cellulose cell walls) or act as energy stores (starch and glycogen).

Carbon dioxide in air enters the leaf through the stomata and diffuses through the spongy mesophyll layer to the palisade cells, through their thin cellulose cell walls, and then through the chloroplast envelope into the stroma.

The fixation of carbon dioxide in the stroma maintains a concentration gradient that aids this diffusion.

CO2 that is a by product of respiration in plant cells may also be used for this stage of photosynthesis.

The series of reactions whereby carbon dioxide is converted to organic molecules is called the Calvin cycle. 

1. Carbon dioxide combines with a carbon dioxide acceptor, a 5 carbon compound called ribulose bisphosphate (RuBP). This reaction is catalysed by the enzyme RuBisCO (ribulose bisphosphate carboxylase-oxgenase).

2. RuBP, by accepting the carboxyl (COO-) group, becomes carboxylated, forming an unstable intermediate 6 carbon compound that immediately breaks down.

3. The product of this reaction is 2 molecules of a 3 carbon compound, GP (glycerate-3-phosphate). The carbon dioxide has now been fixed.

4. GP is then reduced, using hydrogens from the reduced NADP made during the light-dependent stage, to triose phosphate (TP). Energy from ATP, also made during the light-dependent stage, is used at this stage at the rate of 2 molecules of ATP for every molecule of carbon dioxide fixed during stage 3.

5. In 10 of every 12 TP molecules, the atoms are rearranged to regenerate 6 molecules of RuBP. This process requires phosphate groups. Chloroplasts contain only low levels of RuBP, as it continually being converted to GP, but is also continually being regenerated. The remaining 2 molecules of TP are the product.

ATP and reduced NADP, formed from the light dependent stage, are continuously needed for the Calvin cycle to run.

During the light-dependent stage, hydrogen ions are pumped from the stroma into the thylakoid spaces, raising the pH to 8, which is optimum for the enzyme RuBisCO.

RuBisCO is also activated by the presence of extra ATP in the stroma.

In daylight, the concentration of magnesium ions increases in the stroma. These ions attach to the active site of RuBisCO, acting as cofactors to activate it.

The ferredoxin that is reduced by electrons from PSI activates enzymes involved in the reactions of the Calvin Cycle.

Some TP molecules are used to synthesise organic compounds, for example:

• Some glucose is converted to sucrose, some to starch and some to cellulose.
• Some TP is used to synthesise amino acids, fatty acids and glycerol.

The rest of the TP is recycled to regenerate the supply of RuBP.

5 molecules of the 3 carbon compound TP interact to form 3 molecules of the 5 carbon compound RuBP.  

The factors that affect the rate of photosynthesis operate simultaneously.

These factors include the raw materials (carbon dioxide and water) as well as the energy source (light intensity), plus availability of chlorophyll, electron carriers and the relevant enzymes.

Other factors, including temperature and turgidity of the cells, are also important.

At any given moment, the rate of a metabolic process that depends on a number of factors is limited by the factor that is present as its least favourable (lowest) level.

Light provides the energy to power the first stage of photosynthesis and produce ATP and reduced NADP needed for the next stage.

Light also causes stomata to open so that gaseous exchange can occur.

When stomata are open transpiration also occurs, and this leads to uptake of water and its delivery to leaves.

At a constant favourable temperature and constant suitable carbon dioxide concentration, light intensity is the limiting factor.

• When light intensity is low, the rate of photosynthesis is low.
• As light intensity increases, the rate of photosynthesis increases.

At a certain point, even when light intensity increases, the rate of photosynthesis does not increase. Now another factor other than light intensity is the limiting process.

1. GP cannot be reduced to TP.

2. TP levels fall and GP builds up.

3. If TP levels fall, RuBP cannot be regenerated.

The levels of CO2 in the atmosphere and in aquatic habitats are high enough that carbon dioxide is not usually a limiting factor.

If the concentration of carbon dioxide falls below 0.01%.

1. RuBP cannot accept it, and accumulates.

2. GP cannot be made.

3. Therefore, TP cannot be made.

The Calvin cycle involves many enzyme catalysed reactions and therefore is sensitive to temperature. The effects of changing temperature on the Calvin cycle are:

• From low temperatures of 20-30°C, if plants have enough water and carbon dioxide and a sufficient light intensity, the rate of photosynthesis increases as temperature increases.

• At temperatures above 30°C, for most plants, growth rates may reduce due to photorespiration: oxygen competes with carbon dioxide for the enzyme RuBisCO's active site. This reduces the amount of CO2 being accepted by RuBP and subsequently reduces the quantity of GP and therefore TP being produced, whilst initially causing an accumulation of RuBP. However, due to lack of TP, RuBP cannot be regenerated.

• At temperatures above 45°C, enzymes involved in photosynthesis may be denatured. This would reduce the concentrations of GP and TP, and eventually RuBP as it could not be regenerated due to lack of TP.

If a plant has access to sufficient water in the soil, then the transpiration stream has a cooling effect on the plant. The water passing up the xylem to the leaves also keeps plant cells turgid  so they can function. Turgid guard cells keep the stomata open for gaseous exchange.

If there is not enough water is available to the plant (water stress):

• The roots are unable to take up enough water to replace that lost via transpiration.
• Cells lose water and become plasmolysed.
• Plant roots produce abscisic acid that, when translocated to leaves, causes stomata to close, reducing gaseous exchange.
• Tissues become flaccid and leaves wilt
• The rate of photosynthesis greatly reduces.

There are many ways to measure the rate of photosynthesis, including the rate of uptake of raw materials, such as carbon dioxide, or the rate of production of the by-product, oxygen. In each case, to measure the rate we need to calculate the quantity taken up or produced per unit time.

In school labatories, the rate of photosynthesis is often found by measuing the volume of oxygen produced per minute by an aquatic plant. There are limitations with this method because:

• some of the oxygen produced by the plant will be used for its respiration
• there may be some dissolved nitrogen in the gas collected.

However, the same apparatus can be adapted and used to measure the effects of light intensity, temperature or carbon dioxide availability on the rate of photosynthesis.

Also known as an Audus microburette. It is set up so that it is air tight and there are no air bubbles in the capillary tubing. Gas given off by the plant, over a known period of time, collects in the flared end of the capilary tube. As the experimenter manipulates the syrine, the gas bubble can be moved into part of the capillary tube against the scale and its length measured. If the radius of the capillary tube bore is known, then this length can be converted to volume. Volume of gas collected = length of bubble x Pi^2. 

• Wave length of light - different coloured filters
• Light intensity - distance of lamp
• Temperature - water baths
• Carbon dioxide concentration - sodium hydrogen carbonate/pond water solution