Journal of Energy Bioscience 2025, Vol.16, No.5, 238-247 http://bioscipublisher.com/index.php/jeb 241 3.4 Stress tolerance engineering To maintain high yields on marginal land, C4 crops need to have stronger stress resistance. By overexpressing antioxidant enzymes, osmotic regulatory substances (such as proline, betaine), heat shock proteins (HSPs), and regulating some transcription factors and mirnas related to stress response, these crops can be made more drought-tolerant, heat-tolerant and salt-tolerant (Nowicka et al., 2018; Yadav and Mishra, 2020; Yadav et al., 2020; Nazari et al., 2024). In addition, C4 photosynthetic genes themselves, such as PEPC and PPDK, can also show the effect of enhancing stress resistance when heterologous expressed. 4 Case Study: Genetic Engineering in C4 Biofuel Crops 4.1 Example: sugarcane genetic engineering for enhanced sucrose and biomass accumulation 4.1.1 Overexpression of PEP carboxylase and rubisco activase Sugarcane is an important fuel and sugar crop worldwide. In recent years, many researchers have used genetic engineering to modify the key photosynthetic enzymes of sugarcane. Through a series of experiments, researchers ultimately discovered that overexpression of genes such as phosphoenolpyruvate carboxylase (PEPC) and Rubisco activase can enhance CO2 fixation and carbon assimilation, effectively increasing the sucrose content and biomass in sugarcane. Regulating genes related to sucrose metabolism, carbon allocation and stem maturation using RNA interference and gene editing is also an effective measure to increase sugar production and biomass (Budeguer et al., 2021; Nazari et al., 2024; Brant et al., 2025). 4.1.2 Results: improved photosynthetic rate, higher sucrose content, increased biomass yield These genetic engineering measures have brought about obvious improvements. The photosynthetic rate of genetically modified sugarcane is higher, and both the sucrose content and biomass yield have increased. For example, sugarcane with overexpression of sucrose phosphosynthase (SPS) not only had significantly increased sucrose content in leaves and stems, but also had better plant height and stem number than the control (Anur et al., 2020; Brant et al., 2025). Furthermore, genetic engineering methods that regulate stem maturity and carbon allocation have further promoted biomass accumulation (Budeguer et al., 2021). 4.2 Example: maize engineering for improved nitrogen use efficiency 4.2.1 Altered expression of GS/GOGAT cycle enzymes Corn is the model plant of C4 crops, and its nitrogen use efficiency (NUE) directly affects the yield. Nitrogen assimilation and reuse can be enhanced by regulating the expression of key enzymes such as glutamine synthase (GS) and glutamine-2-ketoglutarate aminotransferase (GOGAT) (Lebedev et al., 2021; Liu et al., 2021; Fortunato et al., 2023; Zheng et al., 2025). Meanwhile, studies have found that identifying and overexpressing genes related to NUE (such as ZmNRL1) can also improve the growth performance of maize when nitrogen is insufficient. 4.2.2 Results: enhanced growth under nitrogen-limited conditions Regulation and modification can enable corn to maintain a high growth rate and yield in a low-nitrogen environment (Lebedev et al., 2021; Fortunato et al., 2023). Overexpression of ZmNRL1 can increase the nitrogen content and chlorophyll level in plants, enhance the tolerance of plants to nitrogen stress, and ultimately achieve higher biomass and yield (Zheng et al., 2025). Optimizing the GS/GOGAT cycle can achieve the goal of improving nitrogen assimilation efficiency and overall productivity (Liu et al., 2021). 4.3 Lessons learned and implications for future biofuel crop engineering Based on the above two cases, it can be concluded that the genetic engineering modification of key enzymes for photosynthesis and nitrogen metabolism has a positive effect on increasing the biomass, sugar production and resource utilization efficiency of C4 crops (Anur et al., 2020; Lebedev et al., 2021; Fortunato et al., 2023; Nazari et al., 2024; Brant et al., 2025; Zheng et al., 2025). However, Budeguer et al. (2021) pointed out that there are still some technical problems and bottlenecks: the genomes of polyploid crops such as sugarcane are relatively complex, the transgenic efficiency is low, and the stability of traits is insufficient. In the future, multiple strategies such as multi-gene editing, transcription factor regulation, and miRNA targeting need to be combined to jointly enhance photosynthetic efficiency, metabolic distribution, and stress resistance (Ahmad and Ming, 2024) (Figure 2).
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