Journal of Energy Bioscience 2025, Vol.16, No.4, 205-215 http://bioscipublisher.com/index.php/jeb 209 genes (PEPC, NADP-ME, MDH, PPDK) are located in different chromosome arms. The expression of these genes varies greatly at different growth stages of wheat plants and among different wheat varieties (Bachir et al., 2017; Daoura et al., 2018; Chen et al., 2023). The evolutionary process of C4 photosynthesis involves multiple procedures such as gene replication, functional differentiation, and changes in expression patterns, which increases the difficulty of gene manipulation. To achieve the engineering of C4, it is necessary to comprehensively understand and precisely regulate these gene networks in advance (Chen et al., 2023; Raturi et al., 2024). 5.2 Coordinated expression of multi-gene networks The C4 photosynthetic mechanism requires the coordinated expression of multiple genes in time and space, especially the division of labor between mesophyll cells and vascular bundle sheath cells (Schuler et al., 2016; Cui, 2021; Chen et al., 2023; Prasanna et al., 2025). At present, although some C4 enzymes can be efficiently expressed in C3 crops, it is still difficult to simultaneously express all key enzymes in the correct cell type and subcellular location (Schuler et al., 2016; Ermakova et al., 2020; Cui, 2021). Factors such as the selection of gene promoters, the combination of regulatory elements, the control of transgenic insertion positions and expression levels can also affect whether the C4 pathway can function normally (Ermakova et al., 2020; Chen et al., 2023). 5.3 Anatomical constraints The efficiency of C4 photosynthesis is very high, mainly relying on the "Kranz" structure. This special structure enables mesophyll cells and vascular bundle sheath cells to be closely arranged and functionally differentiated (Schuler et al., 2016; Cui, 2021; Mukundan et al., 2024; Prasanna et al., 2025) (Figure 2). As wheat is a C3 crop, it naturally lacks this structure in its body, making it difficult to form an effective CO₂ concentration mechanism. Although anatomical and functional differentiation structures similar to C4 (such as the "Bose" structure) have emerged during wheat grain development, it remains difficult to reconstruct the Kranz structure in leaves (Rangan et al., 2016; Cui, 2021; Rangan et al., 2024). At present, scientists have not yet fully grasped the genetic regulatory mechanism of the Kranz structure, which to some extent restricts the progress of C4 engineering. Figure 2 Anatomy and physiology of C3 and C4 plants (Adopted from Mukundan et al., 2024) Note: A Histology of NAD-ME dorsiventral C4 leaf showing the mestome (a non-chlorophyllous layer of tissue between the bundle sheath and vascular bundle) along with Kranz features B Histology of isobilateral C4 leaf showing the Kranz features C C3 pathway (Calvin cycle) showing the enzyme, substrate and carbon assimilation D C4 pathway (Hatch and Slack pathway) showing the enzyme, substrate and carbon assimilation along with decarboxylation enzymes (Adopted from Mukundan et al., 2024) 5.4 Metabolic balance and trade-offs Introducing the C4 pathway into C3 crops will have a significant impact on the original C3 metabolic network, which may bring about new metabolic bottlenecks or side effects (Cui, 2021; Prasanna et al., 2025). For instance, the high-level expression of C4 enzymes needs to be matched with energy supply, reducing power distribution, and carbon flow balance; otherwise, it may inhibit growth or waste resources (Ermakova et al., 2020; Cui, 2021; Prasanna et al., 2025). Furthermore, the synergistic patterns of the C4 and C3 pathways in different tissues and at different developmental stages remain unclear and require further optimization through systems biology and metabolic modeling (Schuler et al., 2016; Cui, 2021; Prasanna et al., 2025).
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