As the human population is at its increasing pace, the crop yield is reaching a plateau. The major focus in this regard will be increasing flux yield through C-3 cycle. The vast variety of plant species fixes atmospheric carbon dioxide using the enzyme RuBisCO in Calvin-Benson cycle. The oxygenation reaction directs the flow of carbon through the photorespiratory pathway and this results in loss of 25-30% of the fixed carbon. The oxygenation reaction can be enhanced by temperature, drought and other environmental factors. Thus, the reduction in Rubisco-oxygenase reaction increases the carbon assimilation significantly. Thats why, currently scientists are trying their best to genetically engineer the crop plant so as to have more efficient Rubisco.

Ribulose-1,5-Bisphosphate Carboxylase Oxygenase (RuBisCO) is an enzyme involved in the Calvin cycle that catalyzes the first major step of carbon fixation. It catalyzes either the carboxylation or the oxygenation of Ribulose-1,5-Bisphosphate (RuBP) with carbon dioxide or oxygen. It is known as the most abundant protein on Earth. The enzyme usually consists of the large and small chain subunits. The large chain is a part of chloroplast DNA molecule and there is several small related small chain genes in the nucleus of the plant cell. The small chains are imported to the stromal compartment of chloroplast from the cytosol by crossing the outer chloroplast membrane. The enzymatically active substrate binding sites are located in the large chains that form dimer in which amino acid from each large chain contribute to the binding sites. Magnesium ions are required for the enzymatic activity. The factors such as, temperature, concentration, pH of the stroma cells, and the level of carbon dioxide affects the enzymatic activity of the RuBisCO enzyme.

The main reason for the limitation of carbon assimilation in plants using the C3 cycle is due to the catalytic activity of the enzyme itself. In many studies, it has been found that there is a possibility of other enzymes being involved in determining the rates of carbon flux through the C3 cycle. To undertake metabolic control analysis of a pathway, it is necessary to be able to reduce specifically the amount of an individual enzyme in that pathway; the effect of this reduction on flux can then be compared to the control. The flux control coefficient can vary from 0 to 1. One basic approach between this approach and that based on the kinetics of individual enzymes is that the metabolic control analysis allows for all enzymes in a pathway to share control of flux in that pathway. The flux control, value for any single enzyme is not constant and can change depending on the conditions under which the analysis was carried out. Transgenic plants with reduction in Rubisco protein levels produced using an antisense construct were used to assess the relative contribution that Rubisco imposed on carbon flux. This approach has demonstrated clearly that the limitation imposed by Rubisco on C3 carbon fixation is greater in light and optimum temperature. In contrast, as expected this is reduced in plants grown in elevated carbon dioxide. These transgenic studies showed that Rubisco does not limit the C3 cycle in all conditions and that enzyme of the regeneration phase of the cycle also play a role in determining the rate of photosynthesis.

Calvin cycle is better known as reductive pentose phosphate cycle or C3 cycle. This is a series of biochemical redox reactions that take place in the stroma of chloroplast in photosynthetic organisms. It is often referred to as dark reaction. There are two stages of photosynthesis- light dependent and light independent reactions. The light reaction captures the energy of light and uses it to make the energy storage molecules ATP and NADPH. The dark reaction uses energy from short lived electronically excited carriers to convert carbon dioxide and water to organic compounds.

It has become a greatest challenge to reduce the rate of global climate induce by the growing levels of greenhouse gas. Subsequently, with the efforts implemented on reducing the level of poisonous gases from the anthropogenic sources, researchers are exploring the technology that can play an important role in effectively managing the long term risks of climate change. The scientists are focused on the evolution of RuBisCO variants with improved kinetic and biophysical properties that could enable the plants to use and convert carbon dioxide more efficiently. Presently, researchers are trying to insert the mutant RuBisCO genes into the bacteria and screen out the most efficient enzyme. Naturally, E.Coli do not carry the RuBisCO enzyme and contribute to the carbon sequestration from the atmosphere. The researchers thus, isolated the genes encoding RuBisCO and a helper enzyme from the photosynthetic bacteria and added them to the bacteria. Such genetically modified E.Coli were able to fix and convert carbon dioxide into the consumable energy when the other nutrients were withheld and the bacteria relied on RuBisCO and carbon dioxide to survive under theses stringent conditions. Subsequently, the gene was randomly mutated and these mutant genes were inserted into the E.Coli. The fastest growing strains carried the mutated RuBisCO genes that produced a larger quantity of the enzyme, leading to faster assimilation of carbon dioxide gas. One avenue is to introduce the enzyme RuBisCO variants alonwith the red algae into the plants. This is expected to improve the photosynthetic efficiency of crop plants, although possible negative impacts have yet to be studied. The advancement in this area includes the replacement of the tobacco enzyme with that of the purple photosynthetic bacterium. The first species of RuBisCO to be successfully cloned and expressed as an active enzyme with identical kinetic characteristics to the authentic protein was from Rhodospirullum rubrum.
The oxygenase reaction of RuBisCO and subsequent photorespiratory pathway are clear targets for the improvement of the C3 cycle. There have been efforts made on re-engineering the RuBisCO protein using site directed mutagenesis to increase the carboxylase relative to the oxygenase reaction.

Recently, it has been shown that it is possible to form active RuBisCO in vitro using unfolded large subunits with the chaperon proteins. This work paves the way for the in-vitro analysis of mutant and has the potential to allow screening for improvements in the RuBisCO catalytic parameters of genetically engineered mutants. The variations in the properties would indicate that transferring RuBisCO from these species into the plant could substantially increase the rate of carbon dioxide assimilation. The prokaryotic carboxysome provides a micro compartment in the cell that transports and fixes carbon dioxide acting as a carbon dioxide concentration mechanism.

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Geetanjali Murari
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