Overview:
In C4 plants, the carboxylation process occurs twice in order to efficiently fix carbon dioxide and minimize photorespiration. This process involves spatial separation of initial carbon fixation and the Calvin cycle. Let's understand it in detail.

1. Initial Carbon Fixation:
- In C4 plants, the first step of carbon fixation occurs in the mesophyll cells, which have abundant chloroplasts.
- Here, the enzyme phosphoenolpyruvate carboxylase (PEP carboxylase) carboxylates phosphoenolpyruvate (PEP) using carbon dioxide to form oxaloacetate (OAA).
- This enzyme has a higher affinity for carbon dioxide than rubisco, the enzyme responsible for carbon fixation in C3 plants.

2. Formation of Four-Carbon Compound:
- The formed OAA is then converted into malate or aspartate, which are four-carbon compounds.
- This conversion occurs in the mesophyll cells, and the four-carbon compounds are transported into bundle sheath cells through plasmodesmata.

3. Release of Carbon Dioxide:
- In bundle sheath cells, the four-carbon compounds are decarboxylated, releasing carbon dioxide.
- This decarboxylation is catalyzed by the enzyme malic enzyme or NADP-malic enzyme.
- The released carbon dioxide is then available for the Calvin cycle.

4. Calvin Cycle:
- The carbon dioxide released in bundle sheath cells enters the Calvin cycle, which occurs in the chloroplasts of bundle sheath cells.
- The Calvin cycle uses the enzyme rubisco to fix carbon dioxide into organic compounds, eventually producing glucose.

Benefits of Double Carboxylation:
- The spatial separation of initial carbon fixation and the Calvin cycle allows C4 plants to minimize photorespiration.
- Photorespiration occurs when rubisco fixes oxygen instead of carbon dioxide, leading to wasteful processes.
- By concentrating carbon dioxide in bundle sheath cells, C4 plants effectively minimize the oxygen concentration and maximize carbon fixation.
- This mechanism is especially advantageous in hot and dry environments, where C3 plants may experience higher rates of photorespiration.

In conclusion, the carboxylation process occurs twice in C4 plants. The initial carbon fixation occurs in mesophyll cells, followed by the formation of four-carbon compounds in mesophyll cells and their transport to bundle sheath cells. In bundle sheath cells, the carbon dioxide is released through decarboxylation and enters the Calvin cycle for further carbon fixation. This double carboxylation process allows C4 plants to efficiently fix carbon dioxide and minimize photorespiration.