# The light-independent stage of photosynthesis

The **light-independent stage** of photosynthesis, also known as the **Calvin cycle**, is the second major stage of photosynthesis.

* **Location**: This stage takes place in the **stroma** of the chloroplasts. The stroma is a thick fluid or gel-like substance that fills the space within the inner membrane and surrounds the thylakoids. It contains enzymes, sugars, and organic acids.
* **Reliance on Light-Dependent Stage Products**: The light-independent stage **does not directly use light energy**. However, it is entirely **dependent on the products of the light-dependent stage**: **ATP** (adenosine triphosphate) and **reduced NADP**. ATP supplies the energy, and reduced NADP supplies the hydrogen. In reality, this stage can only continue for a short time after it gets dark because it needs these products.
* **Main Process: The Calvin Cycle (Carbon Fixation)**:
  1. **Carbon Fixation**: Carbon dioxide (CO2) enters the leaf through stomata and diffuses into the stroma. Here, it combines with a 5-carbon compound called **ribulose bisphosphate (RuBP)**. This reaction is **catalysed by the enzyme rubisco** (ribulose bisphosphate carboxylase). It initially forms an unstable 6-carbon compound that quickly breaks down into two molecules of a 3-carbon compound called **glycerate 3-phosphate (GP)** (also known as glycerate phosphate or PGA). This is often called carbon fixation because carbon from CO2 is 'fixed' into an organic molecule.
  2. **Reduction of GP**: The hydrolysis of ATP (from the light-dependent reaction) provides energy to reduce the GP to a 3-carbon compound called **triose phosphate (TP)** (also known as glyceraldehyde 3-phosphate or GALP). This reaction also requires H+ ions, which come from **reduced NADP** (also from the light-dependent reaction). Reduced NADP is then recycled back to NADP.
  3. **Regeneration of RuBP and Product Synthesis**: Most (five out of every six) of the triose phosphate molecules are used to **regenerate RuBP**. This regeneration process also requires **ATP**. The remaining triose phosphate (one-sixth) is converted into **useful organic compounds**. These include:
     * **Carbohydrates**: Hexose sugars (like glucose) are made from two triose phosphate molecules. Larger carbohydrates such as **sucrose, starch, and cellulose** are made by joining hexose sugars together. Starch is stored in starch grains in the stroma.
     * **Lipids**: Synthesised using glycerol (from triose phosphate) and fatty acids (from glycerate 3-phosphate).
     * **Amino acids**: Some are made directly from glycerate 3-phosphate.
* **Summary of Inputs and Outputs**:
  * **Inputs**: Carbon dioxide (CO2), ATP, Reduced NADP.
  * **Outputs**: Organic substances (e.g., glucose, triose phosphate), and the regeneration of RuBP.
* **Rubisco's Role**: Rubisco is noted as one of the **slowest-working enzymes** in the natural world. It is also the most abundant enzyme present in the living world, making up the bulk of all protein in green plants. This abundance is expected because, for photosynthesis to yield useful products, CO2 needs to combine with RuBP, and rubisco catalyzes this crucial first step. Rubisco can also bind with oxygen, leading to **photorespiration**, which is a wasteful reaction that reduces photosynthetic yield, especially in high temperatures and high light intensity.
* **Compensation Point**: Plants carry out both photosynthesis and respiration simultaneously. The **compensation point** for light intensity is the level of light intensity at which the rate of photosynthesis exactly matches the rate of respiration. At this point, oxygen is used as quickly as it is produced, and there is no net exchange of CO2 or O2 with the environment.
* **Experimental Investigation**: The Calvin cycle can be investigated using radioactive carbon-14 (14CO2). By exposing plants to 14CO2 for varying durations and then analyzing the labeled metabolites, the sequence of carbon fixation can be traced.