The Discovery of Dark Reaction
Carbon-14, a radioactive isotope of carbon was served as a tracer to study the details in light independent reactions by Melvin Calvin's team at the Radiochemistry Laboratory of Berkeley University. Green algae were placed in an environment containing Carbon-14 labeled carbon dioxide. The experiment was stopped at different time to kill green algae in boiling ethanol. By analyzing radioactivity in plant samples, Calvin's team was able to determine the sequence of intermediate synthesis.
The C-14 was firstly found on carboxyl group in 3-PGA. The plausible conclusion that a two-carbon compound was involved in biological carbon fixation led research team to keep doing useless work. The exact steps in dark reaction had been a secret until they discovered that pentose was also involved in. Along the right path, researchers identified it is a cyclic process that also called Calvin cycle or C3 pathway. This tough exploration journey lasted about 10 years.
In higher plants, Calvin cycle or dark reaction is the only bio carbon fixation pathway even C4 and CAM plant can't bypass. Because it doesn’t rely on sunlight directly, it gets the name “dark reaction or light-independent reaction”. These terms are inaccurate, since these reactions must also take place in light, only without sunlight direct participation.
Three Steps of Dark Reaction or Calvin Cycle
The light-independent reaction is divided into three main steps in chloroplast stroma: bio carbon fixation or carboxylation, reduction phase, and regeneration of RuBP. A carbon dioxide molecule and a five-carbon saccharide (RuBP) combine into an unstable hexose with the help of enzyme called Rubisco. It quickly breaks down into two 3-phosphoglyceric acid that has a carboxyl group, a phosphate group and a skeleton with three carbons.
The second stage is a reduction and energy storage process. 3-PGA is phosphorylated by one ATP, and then reduced to triose by one NADPH, namely glyceraldehyde 3-phosphate or G3P. Energy is transferred from high-energy compounds ATP and NADPH to triose. The saccharide synthesis is completed at this point. As for how they turn into other saccharide, that is the biochemical reactions occur in chloroplast stroma and cytoplasm. It is no longer part of photosynthesis.
The third stage is RuBP regeneration. Some G3P leaves Calvin cycle, while others remain in cycle. Five trioses (G3P) are converted into three pentoses (RuBP) under the catalysis of enzymes. This is a process of ATP consumption and carbon skeleton rearrangement. Then RuBP enters the next cycle as carbon dioxide acceptor.
The dark reaction fixes only one carbon dioxide molecule each time. Producing one triose requires three Calvin cycles. Each cycle consumes three ATP and two NADPH. All the enzymes for dark reaction are in the stroma of chloroplast. G3P serves as a fundamental building block in the synthesis of glucose, sucrose, and starch. Additionally, it also acts as a precursor for amino acids and fatty acids.