## 3 Results
### Stability of the System during the Simulation Process  
In this study, we calculated the root mean square deviation (RMSD) values to assess the stability of the system and the deviation of different molecules within the system throughout the simulation. The RMSD values provide insights into the equilibrium state of the system and indicate whether it has reached stability. The analysis demonstrates that the RMSD value of the PSBD1 remains relatively constant at around 1.5 A after 30 ns of simulation, indicating system equilibrium. Moreover, the overall RMSD values are below 2 A, indicating minimal structural changes occurring in the system. The red curve represents the variation of (-)-Spirobrassinin's binding site relative to its initial position, showing slight fluctuations. This suggests that the binding site of (-)-Spirobrassinin undergoes subtle adjustments. However, the overall value remains below 1.4 A, suggesting minimal differences in the position of the binding site (Figure 4A). The radius of gyration (Rg) is a parameter used to assess the compactness of a protein structure, with lower values indicating a more compact structure and higher values indicating a more extended or loose structure. An increase in protein compactness is generally associated with improved stability. Our analysis demonstrates that the compactness of the PSBD1 increases upon binding of the (-)-Spirobrassinin, as evidenced by a decrease in Rg values throughout the simulation period. This trend continues until approximately 30 ns, after which the change in compactness becomes more moderate (Figure 4B). Figure 2: Effects of (-)-Spirobrassinin on the enzymatic activities of different target sites. * Indicates significant differences relative to the control (\(p<0.05\)) and each bar graph is the mean \(\pm\) standard error. Lowercase letters indicate significance (\(p<0.05\)). The different symbols and colors in the columns indicate the enzymatic activities data of \(L\). _sativa_ Linn. seedlings after (-)-Spirobrassinin treatments. (**A**), Influence of (-)-Spirobrassinin on ALS activity; (**B**), influence of (-)-Spirobrassinin on DHAD activity; (**C**), influence of (-)-Spirobrassinin on HPPD activity; (**D**), influence of (-)-Spirobrassinin on PPO activity; (**E**), impact of (-)-Spirobrassinin on _C. vulgaris_ growth; and (**F**), influence of (-)-Spirobrassinin on phytohormones content in \(L\). _sativa_ Linn. ABA: abscisic acid; IAA: indole-3-acetic acid; ICA: indole-3-carboxaldehyde; ICAId: indole-3-carboxaldehyde; ME-IAA: Methyl indole-3-acetate; St: striglocalotes; ABA-GE: ABA-glucosyl ester; and GA: gibberellin. Figure 4: Stability during simulation period. (**A**), The trend of the PSBD1’s radius of gyration (Rg) values during the simulation period. (**B**), The trend of the PSBD1’s solvent accessible surface area (SASA) values during the simulation period. (**C**), The trend of RMSD values for the molecules in the system during the simulation period. (**D**), The statistical analysis of the root mean square fluctuation (RMSF) of each amino acid in the PSBD1 during the simulation period. Figure 3: Snapshots from the 50 ns molecular dynamics simulation showing the (-)-Spirobrassinin (shown in green) bound to the PSBD1 (represented by ribbon visualization). (-)-Spirobrassinin and PSBD1 are represented by spheres and cartoon representations, respectively. The variation in the solvent-accessible surface area (SASA) of the protein provides insights into its structural changes and reflects the degree of burial of hydrophobic surfaces. A higher SASA value indicates a larger exposed area of the PSBD1 with fewer buried hydrophobic surfaces, while a smaller SASA value suggests a more compact PSBD1 structure with more buried hydrophobic surfaces. Our analysis indicates that the SASA of the PSBD1 decreases during the simulation period, indicating structural compaction.