Journal of Energy Bioscience 2025, Vol.16, No.5, 238-247 http://bioscipublisher.com/index.php/jeb 240 of the CO2 concentration mechanism, C4 plants can still carry out photosynthesis when the stomata are partially closed, with less water transpiration loss, so they have strong drought resistance (Ghannoum et al., 2010; Ellsworth and Cousins, 2016; Silva-Alvim et al., 2025). The utilization efficiency of nitrogen is also high. C4 plants have a lower demand for RuBisCO than C3 plants, and the nitrogen distribution in their leaves is more reasonable. Therefore, under the same nitrogen input, their CO2 assimilation rate is higher, which can also reduce fertilizer use (Sharwood et al., 2016; Sahoo et al., 2024; Prasanna et al., 2025). Furthermore, C4 crops can still maintain a good yield in environments such as high temperature, strong light, and drought, and can be planted on marginal land, thereby reducing competition with food crops (Monson et al., 2025). 2.3 Limitations and challenges in natural C4 performance. Although C4 crops have great potential in the field of biofuels, they themselves also have some limitations. For instance, their photosynthetic efficiency has not yet reached the theoretical maximum value. The actual efficiency in the field is affected by enzyme activity, stomatal regulation and reaction rate under environmental changes (Verhage, 2021). The adaptability to environmental changes is also limited. Under rapid changes in light or extreme temperatures, the photosynthetic regulation of C4 crops will be delayed, resulting in a decrease in carbon assimilation efficiency (Keller et al., 2021; Lee et al., 2021; Wang et al., 2021; Wang, 2024). The difficulty of genetic improvement is also very high. C4 Photosynthesis depends on complex anatomical structures (Kranz structure) and multi-gene regulation. During genetic engineering modification, problems such as cell-specific expression and metabolic coordination need to be addressed (Coelho et al., 2017; Ermakova et al., 2020; Cui, 2021; Sahoo et al., 2024). Another point is that their adaptability to changes in CO2 concentration is limited. When the concentration of atmospheric CO2 rises, the resource allocation of some C4 crops does not fully keep up with the changes in the modern environment (Pignon and Long, 2020). 3. Genetic Engineering Strategies to Enhance C4 Photosynthesis 3.1 Improving carbon fixation efficiency To enhance the carbon fixation efficiency of C4 plants, it is mainly necessary to improve the expression and activity of key enzymes. By enhancing the functions of specific enzymes such as phosphoenolpyruvate carboxylase (PEPC), pyruvate phosphodikinase (PPDK), and NADP-malate enzyme (NADP-ME) through genetic engineering, the concentration and assimilation efficiency of CO2 can be improved. Meanwhile, Reduce the loss caused by photorespiration (Yadav and Mishra, 2020; Yadav et al., 2020; Nazari et al., 2024). Regulating the expression of carbonic anhydrase (CA) and CO2 channel proteins helps mesophyll cells better acquire and transport CO2. RuBisCO and its activating enzymes can also be modified to increase their activity in vascular bundle sheath cells and further improve carbon assimilation efficiency (Wang et al., 2021). 3.2 Optimizing light harvesting The photosynthetic electron transfer of C4 plants takes place respectively in mesophyll cells and vascular bundle sheath cells. By regulating the expression of certain photosystem proteins (such as the cytochrome b6f complex) through genetic engineering, the light energy between the two types of cells can be rationally allocated, thereby enhancing the overall efficiency. Adjusting the size and composition of the optical capture antenna complex, expanding the spectral range of absorption, and reducing energy loss is also an effective measure to improve the utilization rate of light energy (Von Caemmerer and Furbank, 2016; Wang et al., 2021; Nazari et al., 2024). 3.3 Metabolic engineering for biomass accumulation The final biomass of C4 crops is influenced by multiple metabolic processes such as carbon assimilation, transport and distribution of assimilates, and cell wall synthesis. To enhance the growth rate and yield of C4 crops, researchers typically employ genetic engineering to regulate genes related to the cell cycle, hormone effects (such as auxin and cytokinin), and cell wall synthesis, and then provide assistance with molecular tools like transcription factors and mirnas. In addition to the above methods, enhancing the synthesis and transport of sucrose and allowing more assimilates to be distributed to storage organs can also effectively increase the yield of biofuel raw materials (Cui, 2021; Zafar et al., 2022; Nazari et al., 2024).
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