Computational Molecular Biology 2025, Vol.15, No.6, 282-290 http://bioscipublisher.com/index.php/cmb 287 5.2 Identification of key nodes and bottleneck pathways When some pathways malfunction, the entire metabolic chain is prone to getting stuck. In the simulation analysis, several nodes involved in antioxidant synthesis, hormone generation, and energy regulation were particularly "sensitive", and these areas are very likely to be the metabolic bottlenecks. Once these links are restricted, resources may accumulate or run out. But this is not a bad thing either - precisely because they control the direction of flux, they instead provide a "clue point" for understanding the metabolic adaptation mechanism under salt stress. Of course, these bottlenecks also imply potential vulnerable areas or regulatory Windows, which could be both restrictions and adjustable entry points (Krasensky and Jonak, 2012). 5.3 Prediction of potential metabolic engineering targets and in silico mutant simulations Not all targets are worth taking, but some pathways do hold potential breakthroughs. By simulating the knockout or overexpression of certain key genes, researchers attempt to identify those "lever positions" that can enhance salt tolerance. Several main lines such as photorespiration, antioxidant biosynthesis, and carbohydrate metabolism have been repeatedly proven to be closely related to stress responses (Tong et al., 2025). The targets selected in this way are not only the data in the model, but also the "seed list" for subsequent experimental verification and engineering improvement. The model cannot directly produce salt-tolerant varieties, but it can help us choose the right direction first and avoid detours. 6 Case Study: Modeling of Central Carbon and Amino Acid Metabolism Under Salt Stress 6.1 Experimental validation of metabolic pathways (e.g., GC-MS data support) Not all computational predictions hold water, so experimental verification is particularly crucial. Tools like GC-MS can directly demonstrate the changes in metabolites within plants under salt stress conditions - and these changes often confirm the "reprogrammed" metabolic pathways predicted by the model. For instance, the accumulation of sugars, organic acids and amino acids under stress conditions has been observed in various crops, and barley is no exception (Zhang et al., 2017; Derakhshani et al., 2020). And these changes actually involve the "reconstruction" of core metabolic pathways such as glycolysis and the tricarboxylic acid cycle. By comparing experiments with simulations, researchers gradually clarified which fluxes were regulated and which paths were deflected under stress. 6.2 Network-based analysis of amino acid accumulation (e.g., proline, GABA) Not all amino acids are activated on a large scale under salt stress; some show almost no activity. However, proline and GABA are always indispensable in metabolic analysis and almost consistently rank among the top high-frequency substances. Their roles are far more than just helping to maintain osmotic pressure; they are more like the pivotal nodes in the entire stress response network. Especially the GABA pathway, when the tricarboxylic acid cycle function is compromised, it can always provide a safety net and output energy, and also stabilize the REDOX state within the cell. Interestingly, once there is a problem with the GABA pathway, not only is energy metabolism impaired, but the structure of the cell wall may also have problems (Renault et al., 2013; Che-Othman et al., 2019). Many studies, through network module analysis, have also found that the abnormal accumulation of this amino acid is not a solo effort but is closely linked to the coordinated regulation of the entire metabolic system. 6.3 Simulating the impact of central carbon metabolism adjustment on energy balance and stress adaptation In the face of high salt, plants do not just sit and wait to die. Carbon metabolism is often the first to take action. In the simulated data, a clear trend is evident: glycolysis is enhanced the fastest, while some less conspicuous pathways, such as the GABA bypass and the glyoxylic acid cycle, are rapidly activated. It is not difficult to understand that when the key enzymes of the TCA cycle start to "strike" under high-salt conditions, alternative detour routes become the main force for emergency rescue (Arense et al., 2010; Sharma and Kapoor, 2023). Through these dynamic simulations, it can be seen how plants redistribute carbon flows to ensure the normal supply of ATP. Moreover, the synergy between carbon metabolism and amino acid metabolism is no coincidence.
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