Where photosynthesis happens: the site
The main site is the mesophyll cells of a leaf. Each has many chloroplasts. Inside a chloroplast there are flat sacs called thylakoids. Stacks of them are grana. The fluid around them is the stroma.
- Thylakoid membrane: light reaction (makes ATP, NADPH, O₂).
- Stroma: Calvin cycle (makes sugar). It has the enzymes.
This split of work is called division of labour inside the chloroplast.
Pigments: the light catchers
A pigment is a coloured substance that takes in light. Leaves have four main ones:
- Chlorophyll a (bright blue-green): the main pigment. It sits at the reaction centre.
- Chlorophyll b (yellow-green).
- Xanthophylls (yellow) and carotenoids (yellow-orange).
The last three are accessory pigments. They catch extra colours, pass the energy to chlorophyll a, and protect it from too much light.
Absorption and action spectrum
The absorption spectrum shows how much light of each colour a pigment takes in. The action spectrum shows how fast photosynthesis goes in each colour. Both peak in blue and red. Green is mostly reflected, so leaves look green.
Two phases: light and biosynthetic
- Light reaction (photochemical phase): in thylakoids. Light is caught, water is split (photolysis), O₂ is released, and ATP and NADPH are formed.
- Biosynthetic phase (dark reaction / Calvin cycle): in stroma. It does not use light directly, but it needs the ATP and NADPH that light made. CO₂ is fixed into sugar.
Light-harvesting complexes and photosystems
Hundreds of pigment molecules form an antenna. They send energy to one special chlorophyll a at the reaction centre. PS I has reaction centre P700 (best at 700 nm). PS II has P680. They are named by the order they were found, not the order they work.
The Z-scheme and non-cyclic photophosphorylation
Photophosphorylation means making ATP (adding phosphate to ADP) using light.
- PS II absorbs light. An electron is pushed up to a high energy level and passes to carriers.
- PS II refills its lost electron by splitting water: 2H₂O → 4H⁺ + O₂ + 4e⁻. This is where the oxygen comes from.
- The electron flows downhill through carriers (cytochrome b₆f) to PS I. On the way, energy pumps H⁺.
- PS I absorbs light, the electron is pushed up again and finally reduces NADP⁺ to NADPH.
Drawn on an energy scale this path looks like a sideways Z. Since electrons go one way and never come back, it is non-cyclic. Products: ATP, NADPH, O₂.
Cyclic photophosphorylation
Only PS I works. The excited electron goes to carriers and comes back to PS I. It happens in the stroma lamellae (they lack PS II and NADP reductase), and when light above 680 nm is available. Product: only ATP. No NADPH, no O₂.
Chemiosmosis: how ATP is really made
ATP is made by a gradient of H⁺ (protons) across the thylakoid membrane. The inside (lumen) gets many H⁺ because:
- water is split on the inside, releasing H⁺;
- an electron carrier moves H⁺ from outside to inside;
- NADP reductase removes H⁺ from the stroma side to make NADPH.
H⁺ cannot cross the membrane on its own. It flows out only through ATP synthase. This enzyme has two parts: CF₀ (a channel in the membrane) and CF₁ (a knob in the stroma). The flow changes the shape of CF₁ and ADP + Pi joins to form ATP. Think of water from a dam turning a turbine.
The Calvin cycle (C₃ cycle)
Scientists traced radioactive carbon (¹⁴C) and found that the first stable product is a 3-carbon acid, 3-phosphoglyceric acid (PGA). So plants with only this path are C₃ plants. The cycle has three stages:
- Carboxylation: CO₂ + RuBP (5C) → 2 PGA, by the enzyme RuBisCO, the most common protein on Earth.
- Reduction: PGA is turned into sugar using 2 ATP and 2 NADPH per CO₂.
- Regeneration: RuBP is rebuilt using 1 ATP per CO₂, so the cycle can go on.
For one glucose (6 C) the cycle turns 6 times: 6 CO₂, 18 ATP and 12 NADPH. That is why ATP : NADPH is 3 : 2, and cyclic photophosphorylation helps top up the extra ATP.
Photorespiration
RuBisCO can bind both CO₂ and O₂. When CO₂ is low and O₂ is high (hot day, stomata closed), it binds O₂. RuBP + O₂ gives one PGA and one phosphoglycolate. This path, through chloroplast, peroxisome and mitochondrion, releases CO₂ and uses ATP but makes no sugar and no ATP. It is pure loss. C₃ plants can lose a good part of fixed carbon this way.
The C₄ pathway (Hatch and Slack pathway)
C₄ plants (maize, sugarcane, sorghum, amaranthus) live in hot, dry, bright places. Their leaves have Kranz anatomy: large bundle sheath cells in a ring around each vein, with thick walls, many chloroplasts and no gaps.
- In mesophyll: PEP (3C) + CO₂ → oxaloacetic acid (OAA, 4C) by PEP carboxylase, which does not bind O₂. First stable product is 4C, hence C₄.
- OAA becomes malic or aspartic acid and moves to the bundle sheath.
- There it releases CO₂. CO₂ becomes very high, so RuBisCO works only as a carboxylase. The Calvin cycle runs here.
- The 3C acid goes back to mesophyll and is turned back into PEP (uses ATP).
C₃ vs C₄ at a glance
| Feature | C₃ | C₄ |
|---|---|---|
| First stable product | PGA (3C) | OAA (4C) |
| Kranz anatomy | No | Yes |
| Photorespiration | Yes | Almost none |
| Best temperature | 20–25 °C | 30–40 °C |
| Examples | wheat, rice, mango | maize, sugarcane |
Factors affecting photosynthesis and the law of limiting factors
Rate depends on outside factors (light, CO₂, temperature, water) and inside factors (number and age of leaves, chlorophyll, mesophyll cells, CO₂ inside the leaf).
Blackman's law (1905): when a process depends on many factors, its rate is set by the factor that is closest to its minimum. Increase that one and the rate rises until another factor becomes limiting.
- Light: rate rises with light at low levels; at high light other factors limit. Very strong light can damage chlorophyll.
- CO₂: often the main limiter because air has only about 0.04%. C₃ plants respond to more CO₂ up to high levels; C₄ plants saturate at about 360 µL/L.
- Temperature: dark reaction enzymes need a good temperature. C₄ plants like it hotter.
- Water: its lack closes stomata (less CO₂) and makes leaves wilt, so the effect is mostly indirect.
Try it: see pigments and oxygen at home
- Pigment race: crush a spinach leaf with a little nail-polish remover (acetone) and put a dot on a strip of white chalk or filter paper. Dip the end in the liquid. After 15 minutes you will see bands: yellow-orange (carotenoids) at top, then yellow, blue-green (chlorophyll a) and yellow-green (chlorophyll b).
- Count the bubbles: put a water plant (Hydrilla) in a glass of water under a lamp. Count bubbles per minute at 20 cm and 60 cm. Add a pinch of baking soda (more CO₂) and count again. Predict first, then check with the 3D free-play step.
Key formulas and definitions
- 6CO₂ + 12H₂O → C₆H₁₂O₆ + 6O₂ + 6H₂O (in light, with chlorophyll)
- Photolysis: 2H₂O → 4H⁺ + O₂ + 4e⁻
- Non-cyclic: ATP + NADPH + O₂ · Cyclic: ATP only
- Carboxylation: RuBP + CO₂ → 2 PGA (RuBisCO)
- 1 glucose = 6 turns = 6 CO₂ + 18 ATP + 12 NADPH
- C₄: PEP + CO₂ → OAA (PEP carboxylase)
Worked examples
1. How many turns of the Calvin cycle are needed to make one glucose, and why?
Each turn fixes one CO₂ (one carbon). Glucose has 6 carbons, so 6 turns are needed.
2. Find the ATP and NADPH needed to make 1 glucose.
Per CO₂: 3 ATP and 2 NADPH. For 6 CO₂: 6 × 3 = 18 ATP and 6 × 2 = 12 NADPH.
3. How many ATP and NADPH are needed for 3 molecules of glucose?
3 glucose = 18 CO₂. ATP = 18 × 3 = 54. NADPH = 18 × 2 = 36.
4. How many O₂ molecules come from splitting 12 water molecules? Where does this O₂ come from?
2H₂O gives 1 O₂, so 12 H₂O gives 6 O₂. The O₂ comes from water, not from CO₂ (shown with heavy oxygen ¹⁸O tracer).
5. Non-cyclic flow makes ATP : NADPH in about 1 : 1 but the Calvin cycle needs 3 : 2. For 6 CO₂, how much extra ATP must come from somewhere?
Calvin needs 18 ATP and 12 NADPH. If non-cyclic flow gives 12 NADPH it gives about 12 ATP. Extra = 18 − 12 = 6 ATP, supplied mainly by cyclic photophosphorylation.
6. A plant gets light 80, CO₂ 30 and a good temperature (score 90). Which factor limits it? What happens if light is doubled?
CO₂ (30) is the lowest, so it limits. Doubling light changes nothing because CO₂ is still the smallest. Raising CO₂ would raise the rate until light or temperature becomes the limiter.
7. A C₄ plant spends 2 extra ATP per CO₂ to regenerate PEP. How much ATP does it use per glucose in total?
Calvin: 3 ATP per CO₂. C₄ extra: 2 ATP per CO₂. Total 5 per CO₂. For 6 CO₂: 5 × 6 = 30 ATP (plus 12 NADPH).
Common mistakes
- Saying the O₂ of photosynthesis comes from CO₂. It comes from water, split by light at PS II.
- Thinking the "dark reaction" happens only at night. It needs ATP and NADPH from light, so it runs in the day; it just does not use light directly.
- Thinking PS I works before PS II because of the number. Electrons go PS II → PS I; the names only show which was found first.
- Calling C₄ plants free of the Calvin cycle. C₄ plants also run the Calvin cycle, but in bundle sheath cells.