Photosynthesis in Higher Plants

Photosynthesis is the process by which green plants make their own organic food from carbon dioxide and water using light energy. It matters for two reasons that are easy to state and hard to overstate. First, it is the source of practically all the food on earth, because every food chain begins with a producer. Second, it is the source of the oxygen in the atmosphere. Everything else that lives, including you, is running on borrowed sunlight.

None of this was obvious. For a long time people assumed plants took their food ready-made from the soil. The first real crack in that idea came in 1770, when Joseph Priestley did his bell-jar experiments. He burnt a candle inside a closed glass jar until it went out, and showed that a mouse placed in that same jar suffocated. The air had been spoiled. But when he kept a sprig of mint growing in the jar, the candle could burn again and the mouse could stay alive. Priestley concluded that plants restore to the air whatever burning and breathing animals remove from it. He did not know it was oxygen, and he did not know light had anything to do with it.

That missing piece was supplied by Jan Ingenhousz. Repeating Priestley's experiment in sunlight and in darkness, he found that the purifying effect happened only in light. He then took an aquatic plant and watched what happened around it under bright sunlight: small bubbles formed on the green parts, and only on the green parts. In the dark the bubbling stopped. Those bubbles were oxygen. So light is essential, and only the green parts of a plant can do the job.

Next came the question of what the plant actually makes. In 1854 Julius von Sachs provided evidence that glucose is produced when plants grow, that this glucose is usually stored as starch, and that the green substance of plants sits inside special bodies within the cell, which we now call chloroplasts. He also noticed that starch appears only in the green parts of a leaf, which is why the starch test with iodine is still used in school laboratories to show where photosynthesis has occurred.

A neat piece of work by T. W. Engelmann then told us which colours of light drive the process. He passed sunlight through a prism to spread it into its spectrum, and laid a filament of the green alga Cladophora across the spread-out colours in a suspension of aerobic bacteria. The bacteria, which need oxygen, crowded around the parts of the filament lying in the blue and the red regions. Since bacteria gather where oxygen is being released, and oxygen is released where photosynthesis is fastest, this told him that blue and red light drive photosynthesis best. A graph of the rate of photosynthesis against the wavelength of light is called an action spectrum, and Engelmann's was the first one ever described.

The last big idea came from C. B. van Niel, who studied purple and green sulphur bacteria. These bacteria do a kind of photosynthesis, but they use hydrogen sulphide instead of water, and what they deposit is sulphur, not oxygen. From this he argued that photosynthesis is essentially a light-driven reaction in which a hydrogen donor reduces carbon dioxide, and he wrote the general form as 2H₂A + CO₂ giving 2A + (CH₂O) + H₂O. In green plants the hydrogen donor H₂A is water, so the by-product A must be oxygen. In other words, the oxygen released by a green plant comes from water, not from carbon dioxide. Later experiments in which plants were supplied with water containing a heavy isotope of oxygen confirmed this directly, because the heavy label turned up in the oxygen gas given off.

Putting it together, the correct balanced equation for photosynthesis in a green plant is 6CO₂ + 12H₂O, in the presence of light and chlorophyll, giving C₆H₁₂O₆ + 6H₂O + 6O₂. Notice that twelve water molecules go in and six come out. That is not sloppy bookkeeping. Twelve are split to supply the hydrogen and the oxygen gas, and six are re-formed later in the process, and the equation is written this way precisely to show that the oxygen released came from water.

The main photosynthetic organ of a plant is the leaf, and inside the leaf the work is done by the mesophyll cells, which are packed with chloroplasts. The chloroplasts inside these cells are not scattered at random. They line up along the cell walls, and they can change their orientation so that the flat face is turned towards the light when the light is weak and edge-on when the light is too strong. A leaf also has a large surface area, a thin blade so that gases do not have far to diffuse, and stomata for gas exchange, so its whole design serves this one job.

A chloroplast is bounded by two membranes, an outer and an inner one. The fluid inside is the stroma, and it contains all the enzymes needed to build sugars, together with the chloroplast's own DNA and ribosomes. Suspended in the stroma is a separate membrane system made of flattened sacs called thylakoids. Thylakoids are stacked one on top of another like a pile of coins, and each stack is a granum. The stacks are connected to each other by flat, unstacked sheets called stroma lamellae. The space enclosed inside a thylakoid is the thylakoid lumen, and it is completely separate from the stroma. Remember that separation, because the whole business of ATP synthesis depends on it.

This gives us a clean division of labour. The membranes of the thylakoids carry the pigments and the electron carriers, so this is where light is trapped and where ATP and NADPH are made. That set of reactions is the light reaction, or the photochemical phase. The stroma holds the enzymes that fix carbon dioxide into sugar, so this is where the dark reaction, or biosynthetic phase, happens. The word dark is a poor name and has confused generations of students. The dark reaction does not need darkness and does not stop in the light. It simply does not need light directly. What it needs is the ATP and NADPH that the light reaction supplies, so in practice it runs while the light reaction is running.

Now the pigments. If you grind up a leaf, extract the pigments and separate them by paper chromatography, you do not get one green band but four. They are chlorophyll a, which appears bright or blue green; chlorophyll b, yellow green; xanthophylls, yellow; and carotenoids, yellow to yellow-orange. So the green of a leaf is a mixture, and the yellow and orange pigments are present all along, hidden by the far greater amount of chlorophyll.

Chlorophyll a is the chief pigment. It is the one sitting in the reaction centre, the one that actually hands over an excited electron to start the chain. The other three are called accessory pigments. They absorb light at wavelengths that chlorophyll a absorbs poorly and pass that energy on to chlorophyll a, so they widen the range of light the plant can use. They also protect chlorophyll a from photo-oxidation, that is, from being destroyed by very bright light.

If you measure how strongly chlorophyll a absorbs each wavelength you get its absorption spectrum, and it has two peaks, one in the blue region near 430 nm and one in the red region near 662 nm, with a deep trough in the green. That trough is exactly why leaves look green: green light is largely reflected and transmitted rather than absorbed. When you now compare this absorption spectrum with the action spectrum of photosynthesis, the two curves have a very similar shape, with the highest rates in the blue and red regions. That close match is the evidence that chlorophyll a is the pigment mainly responsible for photosynthesis. The match is not perfect, however, and the difference between the two curves is largely accounted for by the accessory pigments, which absorb light in the regions where chlorophyll a absorbs poorly and pass that energy on.

The pigment molecules in a thylakoid membrane are not floating about singly. They are organised into groups called photosystems, and each photosystem has two parts. Most of the pigment molecules form a light harvesting complex, sometimes called the antenna, whose only job is to catch photons and funnel the energy inwards. At the centre sits a single special pair of chlorophyll a molecules, the reaction centre, which is the molecule that actually loses an electron.

There are two photosystems, and they are distinguished by the wavelength their reaction centre absorbs best. In photosystem I the reaction centre chlorophyll a absorbs maximally at 700 nm, so it is called P700. In photosystem II it absorbs maximally at 680 nm, so it is called P680. A point that trips students up: the numbering reflects the order in which the two photosystems were discovered, not the order in which they work. In the main pathway, photosystem II acts first and photosystem I second.

The two are also in different places on the membrane. Photosystem II sits mainly in the tightly stacked, appressed regions of the grana thylakoids. Photosystem I sits on the stroma lamellae and on the exposed, non-appressed parts of the grana. The stroma lamellae membranes contain neither photosystem II nor the enzyme NADP reductase. That single anatomical fact explains a lot later on.

Now follow the electrons. Light strikes photosystem II. The energy is funnelled to P680, which loses an electron. The electron is picked up by a primary electron acceptor whose position on the membrane faces the stroma, and from there it is passed downhill through a chain of carriers, the electron transport chain: plastoquinone, then the cytochrome b6-f complex, then plastocyanin. As the electron moves down this chain it loses energy, and that energy is used to move protons across the membrane, which is the point of the whole exercise. The electron finally arrives at photosystem I and refills the vacancy in P700.

Meanwhile photosystem I has been absorbing light of its own. P700 loses an excited electron to a second acceptor, one that holds the electron at an even higher energy than the first acceptor did. From there the electron passes through a short chain, reaching ferredoxin, and then the enzyme NADP reductase, which sits on the stroma side of the thylakoid membrane. This enzyme uses two electrons together with protons taken from the stroma to reduce NADP⁺ to NADPH + H⁺.

P680 is now short of an electron and something has to replace it. The replacement comes from water. Associated with photosystem II is a water splitting complex, and it is located on the inner side of the thylakoid membrane, that is, on the lumen side. It breaks water down according to 2H₂O giving 4H⁺ + O₂ + 4e^-. The electrons refill photosystem II, the oxygen is released as the gas we breathe, and the protons are dumped straight into the thylakoid lumen. Note carefully that oxygen evolution is tied to photosystem II, not photosystem I, and that the protons from water splitting end up inside the lumen because that is the side of the membrane where the splitting happens.

The whole route from water to NADPH is called the Z scheme, and the name confuses people who have never seen the graph, so here is what it means in words. Imagine plotting every carrier on a vertical scale of redox potential, with strongly reducing carriers, that is, ones eager to give away electrons, placed near the top and strongly oxidising ones near the bottom. Reading left to right, the electron starts low at P680, jumps sharply upward when light excites it, then slides gradually downward as it travels along the electron transport chain, jumps sharply upward again at P700 when light hits the second photosystem, and finally slides down to NADP⁺. Two steep upward jumps separated by a long downward slope: that traces out the shape of a letter Z lying on its side, and that is all the name means. The important idea is that one photon is not enough. Light has to lift the electron twice, once at each photosystem, to raise it from the very low energy it had in water to the high energy it needs to reduce NADP⁺.

The synthesis of ATP in the presence of light is called photophosphorylation, and there are two versions of it that differ in the route the electrons take.

Non-cyclic photophosphorylation is the pathway described in the Z scheme. Both photosystems take part, the electron starts in water and finishes in NADPH, and because the electron never returns to where it started, the flow is called non-cyclic. Its products are ATP, NADPH and oxygen. This is the main pathway, and it is the one that supplies the Calvin cycle, which needs both ATP and NADPH.

In cyclic photophosphorylation only photosystem I is involved. The excited electron leaves P700, travels through ferredoxin and then, instead of going on to NADP⁺, it is passed back down the electron transport chain and returns to P700. The electron has gone around a closed loop, hence cyclic. Since photosystem II is not involved, no water is split and no oxygen is released, and since the electron never reaches NADP reductase, no NADPH is made. The only product is ATP.

Where does this happen? On the stroma lamellae. Recall that these membranes contain photosystem I but lack photosystem II and lack NADP reductase, so they are physically incapable of doing anything except the cyclic route. Cyclic photophosphorylation also takes over when only light of wavelength beyond 680 nm is available, because such light can excite P700 but not P680. The plant needs the cyclic route because the Calvin cycle uses ATP and NADPH in a fixed ratio of three to two, while non-cyclic flow does not produce them in that ratio. The cyclic pathway makes up the shortfall in ATP.

Now the mechanism itself, which is the chemiosmotic hypothesis. The idea is the same one you meet in respiration: ATP synthesis requires a gradient of protons across a membrane. In a chloroplast, protons pile up inside the thylakoid lumen, so the lumen becomes acidic while the stroma becomes short of protons. Three separate things create that gradient, and you should be able to list all three.

First, water is split on the inner side of the thylakoid membrane, so the protons released by water splitting go directly into the lumen. Second, as electrons move through the carriers, protons are ferried across the membrane. The primary electron acceptor lies towards the outer, stroma-facing side, and it passes its electron not to a plain electron carrier but to a carrier that transports hydrogen. That carrier picks up a proton from the stroma along with the electron, moves across the membrane, and when it hands the electron on to the next carrier on the inner side, the proton is released into the lumen. So every electron passing through drags a proton from stroma to lumen. Third, NADP reductase sits on the stroma side, and when it reduces NADP⁺ to NADPH + H⁺ it takes protons out of the stroma. All three effects push in the same direction: more protons inside, fewer outside.

The gradient is then cashed in by the enzyme ATP synthase, which has two parts. The F0 part is embedded in the thylakoid membrane and forms a channel through it, allowing protons to move back out of the lumen into the stroma by facilitated diffusion. The F1 part sticks out from the membrane on the stroma side. As protons rush back through the F0 channel, the collapse of the gradient releases enough energy to cause a change in shape of the F1 particle, and that conformational change is what joins ADP and inorganic phosphate into ATP. In chloroplasts these two parts are often written as CF0 and CF1 to distinguish them from the mitochondrial enzyme. Because F1 faces the stroma, the ATP appears in the stroma, which is exactly where the Calvin cycle will need it. The NADPH made by NADP reductase, also on the stroma side, is waiting there too. Both are then used up in fixing carbon dioxide, which is the subject of the next section.

The ATP and NADPH made in the light reaction cannot be stored or exported, so the plant spends them at once on building sugar. This happens in the stroma and is called the biosynthetic phase, the dark reaction, or best of all the Calvin cycle, after Melvin Calvin, who worked it out by supplying algae with radioactively labelled carbon dioxide and then checking, after very short intervals, which compound the label had turned up in.

What he found is the fact you must not get wrong. The first stable product of carbon dioxide fixation is a three-carbon acid, 3-phosphoglyceric acid, usually shortened to PGA. Because the first stable product has three carbons, plants that use only this pathway are called C3 plants and the pathway is called the C3 pathway. Most plants you meet, including rice, wheat, potato and most trees, are C3 plants.

The molecule that actually accepts the carbon dioxide is not a three-carbon molecule at all, and this catches people out. The acceptor is ribulose-1,5-bisphosphate, or RuBP, which has five carbons. Carbon dioxide is attached to it by the enzyme ribulose bisphosphate carboxylase-oxygenase, mercifully shortened to RuBisCO. The resulting six-carbon compound is unstable and immediately splits into two molecules of PGA. So one carbon dioxide plus one five-carbon acceptor gives two three-carbon products.

The cycle has three stages. Carboxylation is the step just described, the fixing of carbon dioxide onto RuBP by RuBisCO, and it is the crucial carbon-fixing step of the whole process. Reduction is a pair of steps in which PGA is converted to a sugar, glyceraldehyde 3-phosphate. This uses two ATP for phosphorylation and two NADPH for reduction, per molecule of carbon dioxide fixed. Regeneration is the set of steps that rebuilds RuBP so the cycle can continue, and it needs one more ATP for the final phosphorylation.

Add that up. For every single molecule of carbon dioxide entering the cycle, the plant spends 3 ATP and 2 NADPH. Note that the ratio is three to two, not one to one, and remember it, because it is the reason the plant needs cyclic photophosphorylation to top up its ATP supply. To make one molecule of glucose, which has six carbons, the cycle must turn six times, taking in six molecules of carbon dioxide, and the total cost is 18 ATP and 12 NADPH. Six turns, not one, because the cycle fixes only one carbon per turn.

Regeneration is where most of the chemistry happens even though it gets the least attention. Out of the twelve three-carbon molecules made from six turns, only two are drawn off to build a hexose sugar. The remaining ten are shuffled through a series of three-carbon, four-carbon, five-carbon, six-carbon and seven-carbon intermediates and rearranged into six molecules of the five-carbon acceptor RuBP. The cycle is genuinely a cycle: it must regenerate its own starting material or it stops after one round.

Two more things about RuBisCO are worth knowing. It is the most abundant enzyme on earth, which is not surprising when you consider how many leaves there are and how slow the enzyme is, so plants compensate by making enormous quantities of it. And its full name ends in oxygenase for a real reason: the same active site that binds carbon dioxide can also bind oxygen. Which one it binds depends on the relative concentrations of the two gases around it, because the binding is competitive. When carbon dioxide is plentiful the enzyme behaves as a carboxylase and the Calvin cycle runs normally. When oxygen wins, the plant enters photorespiration, and that is a costly detour we will come to next.

Start with the problem, because the C4 pathway makes no sense unless you see what it solves. RuBisCO can bind either carbon dioxide or oxygen at the same active site, and they compete. In a normal C3 leaf on a hot bright day, the stomata close down to save water, carbon dioxide inside the leaf falls, oxygen from the light reaction builds up, and the ratio shifts in favour of oxygen. When oxygen binds RuBP instead of carbon dioxide, the reaction takes a different course. Instead of two molecules of PGA, RuBP yields just one molecule of phosphoglycerate, which is the three-carbon PGA, and one molecule of phosphoglycolate, a two-carbon compound. Salvaging that two-carbon compound is what we call photorespiration.

Photorespiration is wasteful in a very specific way, and you should be able to state it precisely: there is no synthesis of sugar, no synthesis of ATP and no synthesis of NADPH. On the contrary, the pathway consumes ATP and releases carbon dioxide that the plant had already gone to the trouble of fixing. It is a leak in the system. A C3 plant on a hot dry day can lose a substantial share of its fixed carbon this way.

C4 plants get around this with a two-stage trick. Examples are maize, sugarcane, sorghum and amaranthus, and they are typically plants of hot, dry, tropical conditions. They still have the Calvin cycle; they have simply added a preliminary step in front of it, worked out by Hatch and Slack, which is why the C4 pathway is also called the Hatch and Slack pathway.

The trick depends on a special leaf anatomy called Kranz anatomy. Kranz means wreath, and the name comes from the ring of cells you see in a cross-section. Around every vascular bundle is a tight sheath of large cells, the bundle sheath cells, and around those lie the mesophyll cells. The bundle sheath cells are unusual: they are large, they contain a very large number of chloroplasts, their walls are thick and impervious to gaseous exchange, and there are no intercellular spaces between them. Together these features seal off the interior of the bundle sheath from the air in the leaf.

Now the chemistry. In the mesophyll cells, the primary carbon dioxide acceptor is phosphoenolpyruvate, or PEP, which is a three-carbon molecule. The enzyme that joins carbon dioxide to it is PEP carboxylase, and the product is oxaloacetic acid, or OAA, a four-carbon acid. Because the first stable product has four carbons, these are called C4 plants. The mesophyll cells of a C4 plant do not contain RuBisCO at all, which is a fact worth underlining. The OAA is then converted, still in the mesophyll, into another four-carbon acid, usually malic acid or aspartic acid, and this is what actually travels.

The four-carbon acid moves into the bundle sheath cells, where it is broken down. This releases carbon dioxide, plus a three-carbon molecule, pyruvic acid. The released carbon dioxide now enters the Calvin cycle inside the bundle sheath cells, because that is where RuBisCO is found in a C4 plant. The pyruvic acid travels back to the mesophyll cells, where it is converted into PEP again at the cost of ATP, and the loop is complete.

Why does this defeat photorespiration? Because the four-carbon acids act as a pump, delivering carbon dioxide into a sealed compartment faster than it can escape. The concentration of carbon dioxide around RuBisCO in the bundle sheath is therefore kept very high. With so much carbon dioxide competing against oxygen for the active site, RuBisCO acts as a carboxylase almost all the time, and photorespiration effectively does not happen. Notice that C4 plants do not avoid RuBisCO's flaw by changing the enzyme; they simply keep it in a room full of the right gas.

The pay-off is real. C4 plants tolerate higher temperatures, keep their stomata relatively closed and so lose less water, do not show saturation of photosynthesis even at very high light intensities, and give higher productivity of biomass. The cost is extra ATP, because regenerating PEP from pyruvic acid is an energy-hungry step, so a C4 plant spends more ATP per carbon dioxide fixed than a C3 plant does. In cool, cloudy or shaded conditions that extra cost is not worth paying, which is why C3 plants have not been driven out. And here is the point that most surprises students: in a C4 leaf the Calvin cycle runs only in the bundle sheath cells, a small minority of the leaf's cells, and yet these plants outproduce C3 plants. High local carbon dioxide and zero photorespiration more than make up for the small number of cells doing the work.

The rate of photosynthesis decides how much a plant grows, so anyone who grows crops cares about what controls it. The rate is usually measured as the amount of carbon dioxide taken in or the amount of oxygen given out per unit time.

The factors fall into two groups. Internal factors belong to the plant itself: the number, size and age of the leaves, the arrangement and internal structure of the mesophyll, the number of chloroplasts, the chlorophyll content, and the number and behaviour of the stomata. These depend on the plant's genetic make-up and growth, so they cannot be changed quickly. External factors are the ones in the surroundings: light, carbon dioxide, temperature and water.

Several of these act at once, and that raises an obvious question. If light is plentiful but carbon dioxide is scarce, what fixes the rate? The answer is Blackman's law of limiting factors, put forward by F. F. Blackman in 1905. The law says that when a chemical process is affected by more than one factor, the rate is set by whichever factor is nearest to its minimum value, and that this factor is the one that will change the rate if its own quantity is altered. In plain terms: a chain is only as strong as its weakest link. Increasing any factor other than the limiting one has no effect at all. Once you raise the limiting factor high enough, some other factor becomes the weakest link, and the rate levels off again until that one is raised too.

Light. At low intensity, the rate rises in a straight line as light increases. Beyond a point the curve flattens, and light saturation is reached at about ten per cent of full sunlight. Because ten per cent of full sunlight is not much, light is rarely the limiting factor in nature except for plants growing in shade or on the floor of a dense forest. Very high light intensity does not help further and can even damage the chlorophyll. Remember to separate light intensity from light quality and duration, which matter as well.

Carbon dioxide. This is the factor that most often limits photosynthesis. Its concentration in the atmosphere is very low, between about 0.03 and 0.04 per cent, and raising it up to roughly 0.05 per cent can increase the rate of carbon dioxide fixation. Beyond that, long exposure to higher levels becomes damaging. C3 and C4 plants respond differently. At low light neither group responds to extra carbon dioxide, since light is limiting for both. At high light both respond, but C4 plants reach saturation at around 360 microlitres per litre, while C3 plants keep responding and saturate only beyond about 450 microlitres per litre. So at present-day atmospheric levels carbon dioxide is limiting for C3 plants but not for C4 plants. This is the reason greenhouse growers of crops such as tomato and bell pepper deliberately enrich the air with carbon dioxide and get higher yields.

Temperature. The dark reactions are enzyme-controlled, so they are strongly temperature-dependent. The light reactions are also sensitive to temperature but much less so. C4 plants have a higher temperature optimum and respond well to hot conditions, while C3 plants have a much lower optimum. This also varies with where a plant naturally grows, so tropical plants have a higher optimum than temperate ones, and a plant adapted to its habitat will photosynthesise best in the temperature range of that habitat.

Water. Water is a reactant in photosynthesis, but its effect on the rate is mostly indirect. Water stress makes the stomata close, which cuts off the supply of carbon dioxide to the leaf. Water stress also makes leaves wilt, which reduces the surface area exposed to light and slows down the leaf's metabolic activity in general. So a plant short of water is limited by carbon dioxide before it is ever limited by the water molecules the reaction itself consumes.