which might cause it to act in a similar way to how RuBisCO activase functions in higher plants. By carbon dioxide Since carbon dioxide and oxygen compete at the active site of RuBisCO, carbon fixation by RuBisCO can be enhanced by increasing the carbon dioxide level in the compartment containing RuBisCO (chloroplast stroma). Several times during the evolution of plants, mechanisms have evolved for increasing the level of carbon dioxide in the stroma (see carbon fixation). The use of oxygen as a substrate appears to be a puzzling process, since it seems to throw away captured energy. However, it may be a mechanism for preventing carbohydrate overload during periods of high light flux. This weakness in the enzyme is the cause of photorespiration, such that healthy leaves in bright light may have zero net carbon fixation when the ratio of O2 to available to RuBisCO shifts too far towards oxygen. This phenomenon is primarily temperature-dependent: High temperatures can decrease the concentration of dissolved in the moisture of leaf tissues. This phenomenon is also related to water stress: Since plant leaves are evaporatively cooled, limited water causes high leaf temperatures. plants use the enzyme PEP carboxylase initially, which has a higher affinity for . The process first makes a 4-carbon intermediate compound, which is shuttled into a site of photosynthesis then de-carboxylated, releasing to boost the concentration of , hence the name plants. Crassulacean acid metabolism (CAM) plants keep their stomata closed during the day, which conserves water but prevents the light-independent reactions (a.k.a. the Calvin Cycle) from taking place, since these reactions require to pass by gas exchange through these openings. Evaporation through the upper side of a leaf is prevented by a layer of wax. Genetic engineering Since RuBisCO is often rate-limiting for photosynthesis in plants, it may be possible to improve photosynthetic efficiency by modifying RuBisCO genes in plants to increase catalytic activity and/or decrease oxygenation rates. This could improve sequestration of and be a strategy to increase crop yields. Approaches under investigation include transferring RuBisCO genes from one organism into another organism, engineering Rubisco activase from thermophilic cyanobacteria into temperature sensitive plants, increasing the level of expression of RuBisCO subunits, expressing RuBisCO small chains from the chloroplast DNA, and altering RuBisCO genes to increase specificity for carbon dioxide or otherwise increase the rate of carbon fixation. Mutagenesis in plants In general, site-directed mutagenesis of RuBisCO has been