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Chloroplasts: catching light and turning it into ATP

A chloroplast is a bag of enzymes wrapped round an enormous area of folded membrane, and the split is not decorative. The membranes catch light and build a proton gradient; the fluid around them does the chemistry that gradient pays for. Get the geography right and the rest of the topic follows.

Before this Eukaryotic cell ultrastructure · Chemiosmosis in aerobic respiration · ATP as the immediate energy currency

Before you start

Chlorophyll absorbs green light — that is why leaves are green. It is exactly backwards, and the wording of the mark scheme punishes it. Chlorophyll absorbs strongly in the blue and in the red and takes almost nothing out of the green band in the middle. The green light is reflected and transmitted, which is precisely why it reaches your eye. A leaf is green because green is the light chlorophyll has no use for.

What you should be able to do

Name the parts of a chloroplast and give each one a job it does in photosynthesis.
Explain why a leaf looks green, using the absorption spectrum of chlorophyll.
Say what an action spectrum measures and why it resembles an absorption spectrum.
Separate leaf pigments by chromatography and calculate an Rf value from your own chromatogram.
Describe the light-dependent reactions in order, from a photon to reduced NADP.
Distinguish non-cyclic from cyclic photophosphorylation by their products.

The organelle, and why it is shaped like that

A chloroplast is between two and ten micrometres long, which makes it comfortably visible under a light microscope, and it is wrapped in two membranes rather than one. Inside the envelope sits a thick fluid, the stroma, and running through the stroma is a separate, closed membrane system: flattened sacs called thylakoids, stacked in places into columns called grana and joined between the stacks by single sheets.

Two compartments, two halves of the process. Everything that needs light happens on or across the thylakoid membranes; everything that needs enzymes and a supply of ATP happens in the stroma around them.

The reason for the stacking is surface area, and it is worth being specific about what the area is for. Chlorophyll molecules are held in the thylakoid membrane, so more membrane means more pigment and more light absorbed per chloroplast. The membranes also have to be sealed: the whole light-dependent stage depends on protons being concentrated on one side of them, and a leaky sac would let that gradient drain away as fast as it was built.

Thylakoid: A flattened, membrane-bound sac inside a chloroplast; the membrane holds the pigments, the electron carriers and ATP synthase.
Granum: A stack of thylakoids, giving a large area of membrane within a small volume.
Stroma: The fluid surrounding the thylakoids, containing the enzymes of the light-independent stage, along with DNA and 70S ribosomes.
Photosystem: A cluster of pigment molecules held in a thylakoid membrane, funnelling absorbed energy to a single chlorophyll a molecule at its reaction centre.

That last point about DNA and 70S ribosomes is not padding. It puts chloroplasts alongside mitochondria as the classic evidence for the endosymbiotic origin of eukaryotes, and it earns a mark in questions that ask what a chloroplast has in common with a prokaryote.

Which light actually gets used

Leaves contain several pigments, not one. Chlorophyll a is the one at the reaction centre of every photosystem and the only one that can pass an electron on. Chlorophyll b, the carotenoids and the xanthophylls are accessory pigments: they absorb wavelengths chlorophyll a is poor at, and hand the energy over to it. A plant with only chlorophyll a would waste most of the middle of the spectrum.

The two curves are measured in completely different ways — one with a spectrometer on an extract, one by counting oxygen bubbles from a live plant — and they still land on the same peaks. That agreement is the evidence that these pigments are the ones driving the reaction.

An absorption spectrum is a graph of how much light a pigment absorbs at each wavelength. An action spectrum is a graph of the rate of photosynthesis at each wavelength. They are not the same measurement and questions do sometimes ask you to say so. The point of putting them on the same axes is the argument they make together: photosynthesis is fastest at the wavelengths the pigments absorb best, so those pigments must be what captures the light.

Look at the middle of both curves. Around 550 nm the absorption falls to a fraction of its blue peak, and the rate of photosynthesis falls with it. Green light is neither absorbed much nor much use, so it bounces off, and a leaf under green light photosynthesises slowly — which is a favourite context for a graph-reading question.

Reading the two spectra together

A student shines light of 550 nm on pondweed and counts far fewer bubbles than under light of 440 nm at the same intensity. Explain the result, and explain why the difference is smaller than the difference in absorption by chlorophyll a alone.

At 440 nm the pigments absorb strongly, so more energy is captured, more electrons are excited and more ATP and reduced NADP are made. The light-independent stage is supplied faster, so oxygen is released faster. At 550 nm most of the light passes through or reflects off the leaf without being absorbed at all.

The difference is smaller than chlorophyll a's own absorption suggests because chlorophyll a is not working alone. Carotenoids and xanthophylls absorb in the blue-green and green region where chlorophyll a is weakest, and pass that energy to the reaction centre, so a little photosynthesis still happens at 550 nm.

Notice the shape of that answer. The first paragraph explains the result; the second explains the discrepancy. Two-part questions like this are marked in two parts.

Pulling the pigments apart

You can show that a leaf holds more than one pigment with a strip of chromatography paper. Grind the leaf with a little propanone, spot the extract onto a pencil origin line, let it dry and spot it again — five or six times, so the spot is concentrated but small — then stand the paper in a shallow layer of solvent with the origin line above the solvent surface.

Two details in that method carry marks. The line is drawn in pencil, because ink would dissolve and run up the paper with the pigments. And the origin sits above the solvent, because a spot dipped into the solvent dissolves off the paper instead of travelling up it.

Both distances are measured from the pencil line, and the spot distance is measured to the centre of the spot. Mark the solvent front as soon as you lift the paper out: it is invisible once the paper dries.

Pigments separate because they differ in how strongly they are attracted to the paper and how soluble they are in the solvent. A pigment that is very soluble and holds weakly to the paper travels a long way; one that clings to the paper barely moves. Carotene, a hydrocarbon with no polar groups at all, runs almost to the solvent front. Chlorophyll b, the most polar of the group, lags at the back.

R_f = distance moved by the spot ÷ distance moved by the solventBoth distances measured from the pencil line, in the same units, so R_f has no units and can never exceed 1.

Because the ratio cancels the units, an R_f value is the same whether the solvent ran three centimetres or thirteen, which is what makes it worth quoting. It is only reproducible for a given solvent, though: change the solvent and every value changes, so a table of R_f values always comes with the solvent named.

The light-dependent stage, in the order it happens

Everything in this section takes place on or across a thylakoid membrane. Four things happen, and although textbooks draw them simultaneously, they make far more sense followed in sequence.

Follow the electron, not the light. It is knocked out of chlorophyll, replaced from a water molecule, passed down a chain that pumps protons as it goes, and finally handed with a proton to NADP. Everything else in the drawing is a consequence of that journey.

Photoionisation. A photon is absorbed by the pigments of photosystem II and its energy funnels to the chlorophyll a at the reaction centre. An electron in that molecule is raised to a higher energy level and leaves the molecule altogether. The chlorophyll has lost a negative charge, so it is now positively charged — hence photoionisation, and hence its urgent need for a replacement electron.

Photolysis. The replacement comes from water. An enzyme on the inner face of the thylakoid membrane splits water using light energy: 2H₂O → 4H⁺ + 4e⁻ + O₂. The electrons refill photosystem II, the protons join the growing pool inside the thylakoid space, and the oxygen is surplus. Every molecule of oxygen you have ever breathed came out of a water molecule split like this, not out of carbon dioxide.

The electron transport chain. The excited electron is picked up by a carrier in the membrane and passed from carrier to carrier, losing energy at each transfer. That energy is not wasted: it drives protons from the stroma into the thylakoid space, so the space fills with H⁺ and a steep concentration gradient builds across the membrane.

Chemiosmosis. The membrane is impermeable to protons except at one place — the channel through ATP synthase. Protons flow back out into the stroma through that channel, down their gradient, and the flow drives the enzyme to phosphorylate ADP. Because the energy for it came from light, the process is called photophosphorylation.

At the far end, the electron reaches photosystem I, is re-energised by light absorbed there, and is passed to NADP along with a proton from the stroma. Reduced NADP and ATP are the two things the light-dependent stage exists to produce, and both are released into the stroma, where the Calvin cycle is waiting for them.

Photoionisation: The loss of an electron from a chlorophyll molecule after it absorbs light energy, leaving the molecule positively charged.
Photolysis: The splitting of water using light energy, giving electrons, protons and oxygen.
Chemiosmosis: The movement of protons down a concentration gradient through ATP synthase, driving the synthesis of ATP.
Photophosphorylation: The synthesis of ATP from ADP and inorganic phosphate using energy that originally came from light.

Two routes, two sets of products

The path just described is non-cyclic photophosphorylation: electrons travel from water, through photosystem II, along the chain, through photosystem I and onto NADP, and never come back. It needs photolysis to keep photosystem II supplied, so it produces oxygen, and it produces both ATP and reduced NADP.

There is a second route. In cyclic photophosphorylation only photosystem I is involved. The excited electron is passed to a carrier and then returned to the same photosystem instead of going on to NADP. Protons are still pumped and ATP is still made, but nothing is reduced and no water needs splitting, so there is no oxygen and no reduced NADP.

	Non-cyclic	Cyclic
Photosystems used	PS II and PS I	PS I only
Where the electron ends up	On NADP	Back on PS I
Is water split?	Yes	No
Oxygen produced?	Yes	No
Products	ATP and reduced NADP	ATP only

Cyclic photophosphorylation is a way of making ATP without making anything else, which is useful when a cell needs ATP for a job unrelated to fixing carbon. Guard cells run it hard: opening a stoma means pumping potassium ions in, and pumping costs ATP but does not need reduced NADP.

TRY IT — Explaining an inhibitor

A herbicide binds to a carrier in the electron transport chain between photosystem II and photosystem I, and blocks it. Predict the effect on the amount of oxygen released by a treated plant and on the amount of reduced NADP in its chloroplasts, and explain both predictions.

Check your answer

Oxygen release falls close to zero. Photolysis only happens because photosystem II keeps losing electrons and needs replacements; with the chain blocked, photosystem II fills up with electrons it cannot pass on, so water is no longer split and no oxygen is produced.

Reduced NADP falls too, and for a different reason: the electrons that would have reduced it can no longer reach photosystem I. Whatever reduced NADP was already in the stroma will be used up by the Calvin cycle and not replaced.

Do not stop at 'photosynthesis stops'. The mark is for connecting the block to the two named products, and the oxygen answer needs the extra step through photolysis rather than a straight assertion.

In the exam

Chlorophyll absorbs blue and red and reflects green. Any answer that has chlorophyll absorbing green light has said the opposite of the graph in front of it.
Photolysis splits water. Write the equation with H₂O on the left and be explicit that the oxygen given off comes from water, not from carbon dioxide — questions using isotopes of oxygen test precisely that.
Name the location. 'In the chloroplast' is too vague once a question is worth more than a mark: say thylakoid membrane for the light-dependent stage, stroma for the light-independent stage.
R_f values are ratios, so they have no units and are never greater than 1. If you calculate 1.4 you have divided the wrong way round.
For cyclic photophosphorylation the products are the whole answer: ATP only, no oxygen, no reduced NADP. Two of those three are negatives and all three are marks.
Say what an action spectrum measures if you are asked to compare it with an absorption spectrum. One is a rate, the other is light absorbed; the similarity between them is the evidence, not the definition.

Check yourself

A student illuminates isolated chloroplasts in a buffer containing a blue dye that turns colourless when it is reduced. The dye loses its colour in the light and stays blue in the dark. Explain these observations, and predict what would happen if the thylakoid membranes were first punctured.

Answer

In the light, photosystem II absorbs energy and electrons are lost from chlorophyll. Those electrons pass along the carriers in the thylakoid membrane, and the dye intercepts them — it is acting in place of NADP as the final electron acceptor. Gaining electrons is reduction, so the dye is reduced and loses its colour.

In the dark no electrons are excited, so nothing is passed along the chain, nothing reduces the dye and it stays blue. The reaction needs light directly, which is what makes it part of the light-dependent stage.

Puncturing the thylakoids would leave the dye still losing its colour, because the carriers are in the membrane and can still pass electrons to it. What would stop is ATP synthesis: protons pumped into the thylakoid space would leak straight back out, no gradient could build, and there would be nothing to drive ATP synthase.

That last part is the discriminating one. It separates the electron chain, which needs an intact membrane only as a scaffold, from chemiosmosis, which needs an intact membrane as a barrier.

Questions

Written to the command words the boards use. Try them on paper before opening a scheme: the marks go to points made, not to length.

Question 15 marks

Describe the light-dependent reactions of photosynthesis, in the order in which they happen, from the absorption of a photon to the formation of reduced NADP.

Mark scheme

B1 a photon is absorbed by the pigments of photosystem II and the energy is funnelled to the chlorophyll a at the reaction centre, from which an electron is raised to a higher energy level and lost altogether, leaving the molecule positively charged
B1 the lost electron is replaced by the photolysis of water on the inner face of the thylakoid membrane, which gives protons, electrons and oxygen
B1 the excited electron is passed from carrier to carrier along the thylakoid membrane, losing energy at each transfer
B1 that energy drives protons from the stroma into the thylakoid space, so a steep proton gradient builds across the membrane
B1 protons flow back into the stroma through ATP synthase, driving photophosphorylation, while the electron reaches photosystem I, is re-energised by light and is passed with a proton to NADP, giving reduced NADP

Question 24 marks

Compare non-cyclic photophosphorylation with cyclic photophosphorylation.

Mark scheme

B1 non-cyclic uses photosystem II and photosystem I, whereas cyclic uses photosystem I alone
B1 in non-cyclic the electron ends up on NADP and never returns, whereas in cyclic it is passed to a carrier and returned to the same photosystem
B1 non-cyclic needs photolysis of water to replace the electrons photosystem II loses, so it releases oxygen, whereas cyclic splits no water and releases none
B1 non-cyclic produces both ATP and reduced NADP, whereas cyclic produces ATP only, which suits a job such as pumping potassium ions into a guard cell

Question 33 marks

Explain why a leaf appears green, referring to the absorption spectrum of chlorophyll.

Mark scheme

B1 chlorophyll absorbs strongly in the blue region and in the red region of the spectrum
B1 its absorption falls to a small fraction of that peak in the green band around 550 nm, so green light is barely absorbed
B1 the green light is reflected and transmitted instead, so it is the light that reaches the eye, and a leaf lit only by green light photosynthesises slowly

Question 43 marks

On a chromatogram of leaf pigments the solvent front has run 84 mm from the pencil origin line, and the centre of the carotene spot lies 79 mm from that line. Calculate the Rf value of carotene.

Mark scheme

M1 Rf = distance moved by the spot ÷ distance moved by the solvent, with both distances measured from the pencil line and the spot measured to its centre
M1 79 ÷ 84
A1 Rf = 0.94, a ratio with no units, which can never exceed 1

Question 53 marks

A plant is watered with water in which the oxygen is the heavy isotope oxygen-18, while the carbon dioxide it is given contains only ordinary oxygen. Suggest where the oxygen-18 will first be detected, and justify your answer.

Mark scheme

B1 the oxygen-18 appears in the oxygen gas the plant releases
B1 the oxygen given off in photosynthesis comes from the photolysis of water at the thylakoid membrane, not from carbon dioxide
B1 water is split into protons, electrons and oxygen, so any label carried by the water leaves in the gas; had the oxygen come from carbon dioxide, unlabelled gas would have been released instead

Question 62 marks

State what an absorption spectrum measures, and state what an action spectrum measures.

Mark scheme

B1 an absorption spectrum shows how much light a pigment absorbs at each wavelength
B1 an action spectrum shows the rate of photosynthesis at each wavelength

Worth remembering

Thylakoid membranes do the light-dependent stage; the stroma does the light-independent stage.
Chlorophyll absorbs blue and red, reflects green, and that is why leaves look green.
Photolysis splits water into electrons, protons and oxygen.
Protons are pumped into the thylakoid space and flow back through ATP synthase: that is chemiosmosis.
Non-cyclic gives ATP, reduced NADP and oxygen; cyclic gives ATP alone.

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