## 4 Discussion  
### Response of Leafy Mustard to Chilling Stress  
Under low-temperature stress, the osmotic pressure inside the plant changes, the membrane system is disrupted, and the cell fluid leaks, leading to an imbalance between the inside and outside of the cell. Plants maintain osmotic pressure balance by regulating the content of osmoregulatory substances, reducing the damage caused by low temperature [27; 28]. The accumulation of soluble sugars increases the concentration of cell fluid, decreases the freezing point of the intracellular solution, decreases the content of free water, enhances the water retention ability of cells, prevents the cytoplasmic matrix from freezing and solidifying, and increases the stability of cell membranes [29]. In this study, the soluble sugar content of most mustard cultivars increased under 5 \({}^{\circ}\)C treatment. The soluble sugar content of the different mustard cultivars ranged from 0.17 to 1.42%, with an average value of 0.99%, which was 0.82 times greater than that of the 25 \({}^{\circ}\)C treatment. This finding is similar to the research results of [PERSON] et al. [21] on melon seedlings. Under chilling stress, the hydrolysis of mustard leaves is enhanced, and macromolecular compounds such as \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Index**} & \multicolumn{2}{c}{**SJTKJ**} & \multicolumn{2}{c}{**DX**} \\ \cline{2-5}  & **5 \({}^{\circ}\)C** & **25 \({}^{\circ}\)C** & **5 \({}^{\circ}\)C** & **25 \({}^{\circ}\)C** \\ \hline Chl content mg/g & 1.50 & 1.04 & 0.93 & 0.71 \\ SS content \% & 0.29 & 0.27 & 0.25 & 0.27 \\ Pro content \(\mu\)g/g & 17.89 & 25.84 & 23.90 & 38.14 \\ SOD activity U/g & 288.69 & 99.80 & 257.44 & 143.65 \\ POD activity U/(g-min) & 3068.11 & 3264.39 & 2963.72 & 3776.50 \\ CAT activity U/(g-min) & 558.92 & 225.67 & 607.58 & 422.92 \\ MDA content nmol/g & 1.91 & 1.46 & 1.37 & 1.72 \\ FV/Fm & 0.79 & 0.76 & 0.68 & 0.74 \\ FV\({}^{\prime}\)/Fm\({}^{\prime}\) & 0.72 & 0.72 & 0.63 & 0.66 \\ \(\Phi\)PSII & 0.16 & 0.06 & 0.08 & 0.26 \\ NPQ & 0.43 & 0.22 & 0.23 & 0.44 \\ qP & 0.22 & 0.09 & 0.13 & 0.39 \\ Rfd & 0.73 & 0.39 & 0.40 & 1.01 \\ \hline \hline \end{tabular}
\end{table} 
Table 7: Physiological differences between most chilling-tolerant and least tolerant mustard cultivars. Figure 3: Chlorophyll fluorescence imaging images of most chilling-tolerant and least chilling-tolerant mustard cultivars. Note: The maximum optical quantum efficiency (Fv/Fm) of PSII is 0.6–0.9 from blue to red, and the steady-state fluorescence decay rate (Rfd) is 0.3–1.1 from blue to red. starch and protein are degraded into soluble sugars, which increases the concentration of cell fluid, increases cell osmotic pressure, and enhances chilling tolerance [30]. Proline is an amino acid involved in the composition of proteins and can rapidly accumulate in low-temperature environments to help proteins transport water efficiently, enhance the tolerance of plants to low-temperature stress [31], and alleviate intracellular redox reactions [32]. Studies have shown that proline content is positively correlated with the chilling tolerance of plants under low-temperature stress [33]. In this study, the proline content of most mustard cultivars increased under 5 \({}^{\circ}\)C, and the proline content of different mustard cultivars ranged from 17.01 to 303.53 \(\upmu\)g/g, with a mean value 1.08 times greater than that under 25 \({}^{\circ}\)C. This finding indicates that during low-temperature stress, membrane lipid peroxidation is enhanced in mustard leaves and excessive oxidant production leads to oxidative damage of cellular biomolecules; proline acts as a reactive oxygen species (ROS) scavenger to reduce the oxidative damage and maintain a balanced state between oxidants and antioxidants. In addition, mustard maintains membrane stability and protein structure by accumulating a large amount of proline, which can act as a nonenzymatic antioxidant to scavenge ROS, protect cell membrane structure, and improve plant resilience [34].