The Calvin Cycle (Light-Independent Reactions)

The Calvin cycle (light-independent reactions) takes place in the stroma of the chloroplast, where the enzyme rubisco fixes carbon dioxide onto ribulose bisphosphate (RuBP), and ATP and NADPH from the light-dependent reactions are used to turn the product into triose phosphate, the starting material for glucose. In brief: the cycle has three stages: carbon fixation, reduction and regeneration of RuBP. To make one molecule of triose phosphate (3 carbons), 3 CO₂ molecules are fixed, using 9 ATP and 6 NADPH. Two triose phosphates can form one glucose. Light is not used directly, but the cycle stops in the dark because ATP and NADPH run out.

The Calvin cycle is the pathway by which inorganic carbon enters the living world. This guide explains the three stages, counts the inputs and outputs, describes rubisco and photorespiration, explains the factors that limit the cycle, works through a carbon-counting example and corrects common mistakes.

What is the Calvin cycle?

The Calvin cycle (also called the Calvin-Benson cycle or the C₃ pathway) is the set of reactions that build sugars from carbon dioxide in the stroma. It is called light-independent because none of its reactions require light directly, but it depends on the products of the light-dependent reactions, ATP and NADPH, and several of its enzymes are activated by light. In most plants, therefore, it runs mainly during the day.

What are the three stages?

1. Carbon fixation: the enzyme rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) attaches CO₂ to RuBP, a 5-carbon sugar with two phosphate groups. The unstable 6-carbon product splits immediately into two molecules of 3-phosphoglycerate (3-PGA, also called glycerate 3-phosphate). So each CO₂ fixed gives two 3-carbon molecules.
2. Reduction: each 3-PGA receives a phosphate from ATP and is then reduced by NADPH (which loses electrons and becomes NADP⁺) to triose phosphate (TP, also called glyceraldehyde 3-phosphate, G3P). The energy of ATP and NADPH is thus stored in the carbon compound.
3. Regeneration of RuBP: most of the triose phosphate (5 out of every 6 molecules) is used, in a series of reactions that need more ATP, to regenerate RuBP so that the cycle can continue.

Only 1 out of every 6 triose phosphates produced is a net gain. It leaves the cycle and is used to make glucose and other sugars, starch, cellulose, amino acids and fatty acids. Sucrose is made in the cytosol and transported through the phloem, while starch is stored in the chloroplast.

Inputs and outputs of the cycle

What is rubisco, and what is photorespiration?

Rubisco is the most abundant enzyme on Earth, and it can make up about half of the soluble protein in a leaf. It works rather slowly, at about 3 to 10 reactions per second, so plants need a lot of it. It also has a weakness: it can react with O₂ instead of CO₂. When this happens, the plant produces a toxic 2-carbon compound, and recovering the carbon through photorespiration uses energy, releases CO₂ and wastes a part of the carbon fixed (in the order of 25 percent in C₃ plants in warm conditions). Higher temperatures and low CO₂ inside the leaf make the problem worse, because O₂ becomes relatively more plentiful as stomata close to save water.

What factors limit the Calvin cycle?

• Carbon dioxide concentration: increases the rate until another factor limits it. Present levels in the air (about 0.04 percent) often limit C₃ photosynthesis in bright light.
• Temperature: the cycle depends on enzymes, so the rate rises with temperature and then falls, as enzymes denature. High temperatures also increase photorespiration.
• Light: controls the supply of ATP and NADPH, and activates enzymes.
• Water: water shortage closes stomata, and limits the entry of CO₂.

In an experiment in which light is suddenly turned off, the levels of RuBP fall and those of 3-PGA rise, because ATP and NADPH are no longer available to convert 3-PGA, while rubisco continues to fix CO₂ for a short time. If CO₂ is suddenly removed instead, RuBP accumulates and 3-PGA falls, because there is no CO₂ to react with RuBP. These observations were used by Calvin’s group to identify the order of the compounds.

Worked example: counting carbon atoms

Follow the carbon through three turns of the Calvin cycle.

1. Three turns: 3 CO₂ (3 carbons) combine with 3 RuBP (3 × 5 = 15 carbons). Total: 18 carbons in 3 molecules of the 6-carbon intermediate.
2. Splitting: these form 6 molecules of 3-PGA (6 × 3 = 18 carbons).
3. Reduction: the 6 molecules of 3-PGA become 6 triose phosphates (18 carbons). This uses 6 ATP and 6 NADPH.
4. Output: 1 triose phosphate (3 carbons) leaves the cycle as the net product. The other 5 triose phosphates (15 carbons) are rearranged to regenerate 3 RuBP (15 carbons), using 3 more ATP.
5. Check the totals: carbons in = 3 (from CO₂) + 15 (RuBP) = 18; carbons out = 3 (net triose phosphate) + 15 (RuBP regenerated) = 18. ATP used = 6 + 3 = 9. NADPH used = 6.

These numbers agree with the table: 9 ATP and 6 NADPH for each net triose phosphate. Doing this twice gives 2 triose phosphates (6 carbons), which combine to make 1 glucose, using 18 ATP and 12 NADPH and 6 CO₂ in total. Matching the supply from the light-dependent reactions shows why the two stages depend on each other.

How do C₄ and CAM plants improve carbon fixation?

In hot, dry climates, C₃ plants lose much carbon through photorespiration. C₄ plants, such as maize, sugarcane and sorghum, first fix CO₂ in mesophyll cells using the enzyme PEP carboxylase, which does not react with oxygen, into a 4-carbon acid. The acid moves to bundle-sheath cells, where it releases CO₂ at a high concentration around rubisco, which reduces photorespiration. CAM plants, such as cacti and pineapples, open their stomata at night to take up CO₂ and store it as an acid, and close them by day, when the stored CO₂ is released for the Calvin cycle, which saves water. These pathways cost extra ATP but are advantageous in warm and dry conditions.

How was the cycle discovered?

Melvin Calvin, Andrew Benson and James Bassham worked out the pathway at the University of California, Berkeley, in the late 1940s and 1950s. They supplied algae (Chlorella) with carbon dioxide containing radioactive carbon-14 and stopped the reactions after seconds by dropping the algae into hot alcohol. They used paper chromatography to separate the labeled compounds and found 3-PGA first. Calvin received the 1961 Nobel Prize in Chemistry. Hugo Kortschak and Marshall Hatch and Roger Slack described the C₄ pathway in the 1950s and 1960s.

Which mistakes are common?

Mistake 1: Calling it the “dark reaction” and saying that it occurs at night

It does not need light directly, but it uses ATP and NADPH made in the light, and most plants run it in daylight.

Mistake 2: Saying that the Calvin cycle makes glucose directly

It makes triose phosphate. Two triose phosphates are combined to form glucose and other sugars.

Mistake 3: Forgetting RuBP regeneration

Most of the triose phosphate is used to regenerate RuBP so that the cycle continues.

Mistake 4: Confusing the location

The Calvin cycle occurs in the stroma. The light-dependent reactions occur in the thylakoid membranes.