Photosynthesis

The chloroplast: a structure built for its job

Photosynthesis happens inside the chloroplast, an organelle a few micrometres long that is surrounded by a double membrane called the envelope. The outer membrane is freely permeable to small molecules, but the inner membrane controls what enters and leaves, so that carbon dioxide, phosphate and the products of the Calvin cycle move in and out in a controlled way. Inside the envelope the organelle is filled with the stroma, a fluid containing the enzymes of the light-independent stage, including rubisco, together with looped chloroplast DNA, 70S ribosomes and starch grains.

Suspended in the stroma is a system of flattened, membrane-bound sacs called thylakoids. Where thylakoids lie on top of one another, like a stack of coins, they form a granum, and neighbouring grana are joined by thylakoid membranes that run through the stroma. The membrane of each thylakoid encloses a narrow thylakoid space. The stacking is not decoration: it packs an enormous area of thylakoid membrane into a small volume, and every part of that membrane that carries a photosystem can absorb light.

The two stages of photosynthesis are separated in space, and the structure of the chloroplast is what makes that possible. The thylakoids, which occur in stacks called grana, are the site of the light-dependent stage, and the stroma is the site of the light-independent stage. Keeping them apart lets the chloroplast build up a proton gradient across the thylakoid membrane, and it keeps the enzymes of the Calvin cycle away from the electron carriers that would otherwise interfere with them.

A chloroplast does not work alone either. In a leaf such as that of a tomato plant the upper surface is a transparent epidermis, and beneath it the palisade mesophyll cells are packed with chloroplasts and lie close to the surface, where most of the light arrives. Air spaces below them let carbon dioxide reach the cells, and the xylem in the veins brings water, which is the raw material for the photolysis reaction.

Chloroplast pigments and how chromatography separates them

Light is not absorbed by the thylakoid membrane as a whole but by pigment molecules embedded in it. Chlorophyll a is the primary pigment: it sits at the reaction centre of every photosystem and it is the molecule that loses an electron when it absorbs light energy. Chlorophyll b, carotene and xanthophyll are accessory pigments, grouped in antenna complexes around the reaction centre. They absorb light of wavelengths that chlorophyll a absorbs poorly, and the energy they capture is passed from molecule to molecule until it reaches a chlorophyll a molecule at the reaction centre.

Each pigment has its own absorption spectrum, so between them the pigments of a leaf cover a far wider range of wavelengths than chlorophyll a could alone. None of them absorbs green light well; green light is mostly reflected and transmitted, which is why a leaf looks green in white light. Carotene and xanthophyll also help to protect chlorophyll from damage by dissipating some of the energy when light is very intense.

Chromatography separates the pigments so that they can be identified. A small spot of leaf extract is placed on the origin line of a chromatography strip and the bottom of the strip is dipped into a solvent such as propanone. The solvent moves up the paper as the mobile phase, carrying the pigments with it. How far each pigment travels depends on two things: how soluble it is in the solvent, and how strongly it is adsorbed onto the water held in the paper, which is the stationary phase.

The position of a pigment is recorded as an Rf value: the distance the pigment has moved from the origin divided by the distance the solvent front has moved from the origin, both measured in the same units. An Rf value has no units and can never be greater than 1. To identify a pigment, run a known pigment alongside it on the same strip, or compare your Rf value with a published value obtained using the same solvent and the same type of paper.

Absorption spectra and action spectra: the evidence that pigments drive photosynthesis

An absorption spectrum is a graph of how much light a pigment absorbs at each wavelength. The absorption spectrum of chlorophyll a has two clear peaks, one in the blue-violet region at about 430 nm and one in the red region at about 660 nm, with a deep trough in the green. Carotene and xanthophyll absorb mainly in the blue-violet part of the spectrum, which is why they look orange and yellow: those are the wavelengths they transmit.

An action spectrum is a graph of the rate of photosynthesis at each wavelength of light, and the rate is measured as oxygen production or carbon dioxide uptake. In 1883 Theodor Engelmann obtained an action spectrum by illuminating a filament of the alga Spirogyra with a spectrum and recording where aerobic bacteria gathered, on the assumption that they would collect where oxygen was being produced most rapidly. Modern versions use a suspension of chloroplasts or a piece of pondweed lit by monochromatic light.

The two spectra are closely correlated: photosynthesis is fastest at the wavelengths the chloroplast pigments absorb most strongly, and slowest in the green. That correlation is the evidence that these pigments are the ones that capture the light energy used in photosynthesis. The small amount of photosynthesis that does occur in green light is the contribution of the accessory pigments, which extend the range of wavelengths a leaf can use.

Both spectra are measurable if everything except the wavelength is kept constant. An absorption spectrum can be measured directly by putting a pigment extract in a spectrophotometer and recording the absorbance at each wavelength. For an action spectrum you need a living system, so you illuminate a chloroplast suspension or a length of pondweed with light of known wavelength and intensity, and measure the rate of oxygen production or the time taken for a redox indicator to be reduced.

The light-dependent stage: photoactivation and photophosphorylation

The light-dependent stage takes place on the thylakoid membranes, where the pigments are held in photosystems. In each photosystem, energy absorbed by the antenna pigments is funnelled to a single chlorophyll a molecule at the reaction centre. When that molecule absorbs the energy it loses an electron and becomes positively charged. This is photoactivation, and it is the moment at which light energy becomes chemical energy, because the electron that leaves carries energy with it.

Non-cyclic photophosphorylation uses both photosystem II (PSII) and photosystem I (PSI). Photoactivation of the reaction centre of PSII sends an electron into the electron transport chain, and the chlorophyll a that lost the electron is restored by electrons that come from water: the oxygen-evolving complex, a protein complex on the inner surface of the thylakoid membrane, catalyses the photolysis of water, 2H2O -> 4H+ + 4e- + O2. The electrons from water replace those lost by PSII and the protons are released into the thylakoid space. The electron travels through the carriers of the electron transport chain, losing energy at each step, until it reaches PSI, where light absorbed by PSI activates it a second time and it is finally passed to NADP. NADP is reduced to reduced NADP using protons from the stroma, so non-cyclic photophosphorylation produces ATP, reduced NADP and oxygen.

Cyclic photophosphorylation uses only PSI. The photoactivated electron leaves PSI, passes along the electron transport chain and returns to PSI, so no reduced NADP is made and water is not split, which means no oxygen is released. The only product is ATP. Cyclic photophosphorylation matters because the Calvin cycle does not use ATP and reduced NADP in equal amounts: more ATP is needed than reduced NADP, and the extra ATP is supplied in this way.

Chemiosmosis: how the proton gradient makes ATP

The electrons that leave the reaction centre carry the energy they picked up from light, and they release it in stages as they pass from one carrier of the electron transport chain to the next. The details of the carriers are not required, but what the released energy is used for is: it drives the transfer of protons from the stroma across the thylakoid membrane into the thylakoid space, against their concentration gradient.

Two things keep the gradient steep. The thylakoid space is very narrow, so moving a small number of protons into it makes a large difference to the concentration there, and the thylakoid membrane is impermeable to protons, so they cannot leak back. The protons released inside the thylakoid space by the photolysis of water add to the protons already transferred in.

Protons can return to the stroma only through ATP synthase, a channel protein that is also an enzyme, in the thylakoid membrane. They pass through it down their concentration gradient by facilitated diffusion, and the energy released by their return is used to phosphorylate ADP to ATP. This coupling of proton flow to ATP synthesis is chemiosmosis, and because the proton gradient was built using light energy, the whole process is called photophosphorylation.

You have met the same mechanism in the mitochondrion, where the energy from reduced NAD and FAD is used to pump protons across the inner membrane and the protons then flow back through ATP synthase. The compartments are arranged differently in the two organelles, and the protons come from different places, but the principle is identical: energy from electron transport builds a proton gradient, and the return of protons through ATP synthase makes ATP.

The Calvin cycle: fixing carbon dioxide into triose phosphate

The light-independent stage, better called the Calvin cycle, takes place in the stroma. It does not use light directly, but it depends completely on the light-dependent stage for its energy. Its first stage is carbon fixation: rubisco (ribulose bisphosphate carboxylase) catalyses the combination of a molecule of carbon dioxide with a molecule of ribulose bisphosphate (RuBP), a 5-carbon compound. The unstable 6-carbon product immediately splits into two molecules of glycerate 3-phosphate (GP), a 3-carbon compound.

The second stage is reduction. Each molecule of GP is reduced to triose phosphate (TP), a 3-carbon compound, in reactions that use reduced NADP and ATP from the light-dependent stage. Reduced NADP is the reducing agent and supplies the hydrogen, ATP supplies the energy for the reaction, and both molecules are converted back to their oxidised forms, ready to be made again in the light.

The third stage is regeneration of RuBP, and it is the stage candidates forget. RuBP must be present for carbon fixation to continue, so most of the triose phosphate produced is recycled rather than exported. Five out of every six molecules of TP are used, in reactions that use ATP, to regenerate three molecules of RuBP, and one molecule of TP is the net product. Six turns of the cycle are needed to release enough carbon to make one molecule of glucose.

The intermediates of the cycle are also the starting points for other molecules. GP is used to produce some amino acids, and TP is used to produce carbohydrates, lipids and amino acids. Triose phosphate can be built into hexose, which can be turned into sucrose for transport in the phloem, into starch for storage, or into cellulose for cell walls; it also supplies the glycerol of lipids and the carbon skeletons of amino acids. This is why photosynthesis provides not only the energy of most food chains but the carbon of the organic molecules of nearly every organism.

Limiting factors and the shape of the rate curve

A limiting factor is the factor in shortest supply, and it sets the rate of the process at that moment: increase it and the rate rises, increase anything else and nothing happens. For photosynthesis the factors you must know are light intensity, carbon dioxide concentration and temperature. Only one of them is limiting at any one time, and when you increase it the rate rises only until the next factor becomes limiting. At low light intensity the rate is almost proportional to light intensity, because light is the limiting factor: more photons are captured on the thylakoid membranes, more ATP and reduced NADP are produced, and the Calvin cycle can use more carbon dioxide. As light intensity continues to rise the curve levels off, because light is no longer limiting and something else, usually carbon dioxide concentration or temperature, is now in shortest supply.

Carbon dioxide concentration works in the same way. The rate rises with carbon dioxide concentration because rubisco has more substrate to fix, so more GP and then more TP are produced, and the increased use of ATP and reduced NADP in the stroma pulls the light-dependent stage along faster. The curve levels off when light intensity or temperature becomes the limiting factor instead, so the plateau is reached at a higher carbon dioxide concentration in bright light than in dim light.

Temperature behaves quite differently, because the Calvin cycle is a chain of enzyme-catalysed reactions. Raising the temperature increases the rate up to an optimum, because enzymes and substrates collide more often and with more energy, but above the optimum the rate falls sharply: the enzymes, including rubisco, are denatured, their tertiary structure is destroyed and they can no longer catalyse the reactions. A change in temperature has much less effect on the light-dependent stage itself, because the transfer of energy from light to excited electrons does not need enzyme action, and that is why a temperature curve has a very different shape from a light curve.

In a whole plant there is one further complication. At very low light intensities the rate of photosynthesis can be lower than the rate of respiration, so the plant releases carbon dioxide instead of taking it in. The light intensity at which the two rates are equal is the light compensation point. In bright light, respiration is a small fraction of photosynthesis, so the net uptake of carbon dioxide that you measure is close to the true rate of photosynthesis.

• Light intensity: increases the rate of the light-dependent stage, so the supply of ATP and reduced NADP to the Calvin cycle rises; the curve levels off once another factor is limiting.
• Carbon dioxide concentration: increases the supply of substrate for rubisco, so the rate of carbon fixation rises until light or temperature limits the rate.
• Temperature: increases the rate of the enzyme-catalysed Calvin cycle up to an optimum, then denatures rubisco and the other enzymes so the rate falls sharply.

Investigating the rate of photosynthesis

Two kinds of investigation are used, and they answer different questions. The redox indicators DCPIP and methylene blue are used with a suspension of chloroplasts to study the light-dependent stage on its own. Both dyes are blue when oxidised and become colourless when they are reduced, because they act as artificial electron acceptors, taking the electrons that would otherwise reduce NADP. Chloroplasts are obtained by grinding spinach leaves in an ice-cold, isotonic, buffered solution and filtering the mixture; the suspension is then mixed with the dye and illuminated. The time taken for the blue colour to disappear, or the fall in absorbance measured with a colorimeter, measures how fast the light-dependent reactions are running. Because the dye replaces NADP and no carbon dioxide is added, this is how Robert Hill showed at Cambridge in the 1930s that isolated chloroplasts release oxygen in the light without any carbon dioxide being present.

To use this apparatus to study light intensity, place the tube at different distances from the lamp, keeping the volumes of suspension and dye and the temperature the same. To study wavelength, put coloured filters in front of the lamp and repeat the investigation for each colour, then plot the rate against wavelength to build an action spectrum.

Whole plants, including aquatic plants, are used to measure the overall rate of photosynthesis. A length of Canadian pondweed, Elodea canadensis, is placed in a test tube of water with a lamp at a measured distance. The pondweed releases oxygen, and the bubbles are counted for a fixed time; alternatively the gas is collected in a capillary tube or a gas syringe, or measured with an oxygen electrode connected to a data logger. The rate is recorded as the volume of oxygen released per minute. Light intensity is varied by moving the lamp, carbon dioxide concentration by using different concentrations of sodium hydrogencarbonate solution, and temperature by standing the tube in water baths at different temperatures.

Whichever apparatus you use, the evaluation marks are in the details. Counting bubbles is a crude measure because the bubbles are not all the same size, and some of the oxygen dissolves in the water instead of appearing as a bubble, so the volume is underestimated. The lamp warms the water as it comes closer, and temperature is itself a limiting factor, so a heat screen or a glass tank of water must stand between the lamp and the tube, or the tube must be held in a water bath. The pondweed should be left to acclimatise at each new light intensity for a few minutes before counting begins, because the rate changes over the first minute or two, and every reading should be repeated so that a mean can be calculated. Light intensity should be measured with a light meter rather than recorded as a distance, because intensity falls with the square of the distance from the lamp.