Chloroplasts and the overall reaction of photosynthesisEdexcel A-Level Biology A: Revision notes

Photosynthesis is the process in which plants use light energy to build glucose from carbon dioxide and water, releasing oxygen:

6CO₂ + 6H₂O → C₆H₁₂O₆ + 6O₂

The equation conceals the key ideas. Light energy is used to split the strong bonds in water molecules (photolysis). The hydrogen from the water is stored in a fuel, glucose, by combining it with carbon dioxide, and the oxygen is released into the atmosphere as a waste product. So the oxygen in O₂ comes from water and not from carbon dioxide.

The energy in light is therefore converted to chemical energy in glucose, which is why photosynthesis is described as an endothermic reaction and the basis of almost every food chain.

When algae were given water containing the heavy isotope ¹⁸O and carbon dioxide containing only ¹⁶O, the oxygen gas released contained ¹⁸O. When they were given carbon dioxide containing ¹⁸O and ordinary water, the oxygen released contained no ¹⁸O, and the isotope was found in glucose and in water. This shows that oxygen is released from the splitting of water, while the oxygen atoms of carbon dioxide end up in glucose and water.

Similarly, in the Hill reaction (about 1937) isolated chloroplasts released oxygen in the light when given an electron acceptor, even in the absence of carbon dioxide, which showed that water is the source of the oxygen and that the light-dependent reaction is separate from the fixing of carbon dioxide.

Chloroplasts are discs about 2–10 µm long, found mainly in palisade mesophyll cells. They have:

• A double membrane (the envelope) that controls what enters and leaves.
• Thylakoids: flattened, membrane-bound sacs that contain chlorophyll in photosystems, with electron carriers and ATP synthase in their membranes. The inside of a thylakoid is the thylakoid space.
• Grana (singular granum): stacks of thylakoids, connected to each other by thin membranes called lamellae.
• The stroma: the fluid around the grana containing the enzymes of the light-independent reactions (including rubisco), starch grains, lipid droplets, DNA (circular) and 70S ribosomes.

• Grana give a very large surface area of thylakoid membrane, so many photosystems absorb light and there are many electron carriers and ATP synthase molecules.
• The small thylakoid space allows protons to build up quickly and so form a steep gradient for ATP synthesis.
• The stroma is next to the thylakoids, so ATP and reduced NADP made in the light-dependent reactions pass quickly to the enzymes of the light-independent reactions that make glucose.
• Chloroplast DNA and ribosomes let the chloroplast make some of its own proteins.
• Starch grains store the products of photosynthesis temporarily.
• The envelope controls the movement of substances so that the stroma has a different composition from the cytoplasm.

Isolate chloroplasts by grinding leaves (spinach or lettuce) in an ice-cold, buffered, isotonic solution (for example sucrose), filtering through muslin and centrifuging. The solution is isotonic to prevent the chloroplasts bursting or shrinking, ice-cold to slow enzymes that would damage them, and buffered to keep the pH constant.

Mix the suspension with DCPIP, a blue dye that takes the place of NADP as the electron acceptor. In the light, electrons released from photolysis reduce DCPIP and it changes from blue to colourless. Measure the time taken, or the fall in absorbance with a colorimeter (red filter), at different light intensities.

Controls: a tube in the dark (wrapped in foil), a tube with boiled chloroplasts and a tube with no chloroplasts, all of which stay blue. Keep temperature, volumes and the concentration of DCPIP constant.