Journal of Energy Bioscience 2025, Vol.16, No.4, 205-215 http://bioscipublisher.com/index.php/jeb 208 Systematic understanding and multi-disciplinary collaboration are required: To successfully achieve C4 engineering, it is necessary to have a deep understanding of the photosynthetic mechanisms of C3 and C4, and combine multiple techniques such as synthetic biology, gene editing, and computational modeling (Schuler et al., 2016; Cui, 2021; Prasanna et al., 2025). The Importance of International Cooperation: International cooperation such as the C4 Rice project provides platforms and resources, promoting theoretical research and technological progress (Schuler et al., 2016; Prasanna et al., 2025). 4 Progress in C4 Engineering in Triticum aestivum 4.1 Genomic resources Wheat (Triticum aestivum L.) is a C3 plant, but many homologous genes related to the C4 photosynthetic pathway have been found in its genome. For instance, the homologous genes of key C4 enzymes such as PEPC, NADP-ME, MDH, and PPDK are distributed on different chromosome arms of the three subgenomes A, B, and D of wheat. These genes are all expressed in the flag leaves of wheat and can be detected at different growth stages. The expression levels of these genes vary greatly among different varieties. This provides a rich genetic resource and genetic diversity basis for the C4 engineering of wheat (Rangan et al., 2016; Bachir et al., 2017). 4.2 Gene discovery and functional characterization Researchers have identified C4 photosynthetically related genes in the wheat genome and confirmed that these genes are highly expressed during grain development, especially in the photosynthetic tissues of the caryopsis, such as the cross cell and tubular cell layers. Enzyme activity detection indicated that PEPC, NADP-ME, MDH and PPDK were all active in wheat flag leaves, and their expression levels were closely related to the photosynthetic rate. The unique C4 photosynthetic pathway in wheat grains also provides a molecular basis for its adaptation in high-temperature and arid environments (Rangan et al., 2016; Bachir et al., 2017). 4.3 Transgenic and genome-editing approaches Scientists have adopted transgenic methods on C3 crops such as rice to express C4-related enzymes, thereby enhancing their photosynthetic efficiency and nitrogen utilization efficiency. However, the C4 project in wheat crops is still in its infancy. At present, research on wheat crops mainly focuses on the cloning and expression regulation of the C4 key enzyme gene, as well as the functional verification in different tissues and at different developmental stages. Synthetic biology and gene editing techniques (such as CRISPR/Cas9) can achieve precise regulation and expression of multiple gene modules. However, at the current stage, completely reconstructing the functional pathway of C4 in wheat remains a considerable challenge (Matsuoka et al., 2003; Cui, 2021; Pradhan et al., 2022; Talukder et al., 2024; Prasanna et al., 2025). 4.4 Anatomical engineering To achieve C4 photosynthesis, the expression of corresponding enzymes is required, as well as specific leaf structures (such as Kranz structures), so that the photosynthetic process can be spatially separated. Currently, researchers have observed chloroplast duality similar to that of mesophyll cells and vascular sheath cells in C4 plant leaves in the caryopsis tissue of wheat grains, providing a natural reference for anatomical engineering. However, reforming the Kranz structure in major photosynthetic organs such as wheat leaves remains a major challenge in the C4 project. Future research needs to delve into the molecular mechanisms that control cell differentiation and spatial expression, and combine methods such as directed evolution and artificial selection to promote new breakthroughs in wheat C4 anatomical engineering (Rangan et al., 2016; Schuler et al., 2016; Li et al., 2017; Cui, 2021; Talukder et al., 2024). 5 Technical Challenges 5.1 Genetic complexity In C4 photosynthesis, multiple key enzymes and regulatory elements need to be mobilized, and the genes of these enzymes and regulatory elements are distributed on different chromosomes and subgenomes. In wheat, C4-related
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