## 4 Discussion
### The Molecular Mechanisms of D. officinal Leaves in Response to Salt Stress  
Sugars (such as sucrose, glucose, trehalose, and fructose) with osmotic protection, carbon storage, and ROS scavenging abilities accumulate under salt stress and are related to salt tolerance [26; 27]. As a molecular stabilizer, trehalose may prevent water loss and biomolecules denaturing during dehydration [28]. The accumulation of trehalose plays a role in osmotic protection when plants are exposed to salinity stress [12]. T6P is produced by the binding of a glucose 6-phosphate and a UDP-glucose under the catalysis of trehalose phosphate synthase, and is cleaved into trehalose by trehalose phosphate phosphate [29]. In this study, the content of T6P and trehalose increased under salt stress (Figure 5A and Table S5), suggesting that trehalose may be involved in ROS scavenging. As a ROS scavenger, raffinose and galacitol may mitigate oxidative damage under abiotic stress conditions. For instance, maize/corn (_Zea mays_) _RAFFINOSE SYNTHASE_ (_ZmRAFS_) enhances plant drought tolerance through either the synthesis of more raffinose or by  
Figure 8: The contents of JA, total flavonoids, and anthocyaninidin in leaves under salt stress. (**A**) JA content at different time points under salt stress. (**B**) The contents of total flavonoids in control and salt-stress treatments. (**C**) The contents of anthocyaninidin in control and salt-stress treatments. Statistical significance was calculated using Student’s \(t\)-tests. * \(p<0.05\), ** \(p<0.01\). generating more myo-inositol via hydrolysis of galactinol [30]. Raffinose and myo-inositol accumulated significantly under salt stress (Figure 5A), suggesting that they may enhance the tolerance of _D. officinal_ seedlings to salt stress. AsA, as a strong and active antioxidant, could help plants scavenge ROS to combat environmental stress [31]. Therefore, AsA enhanced the tolerance of _D. officinal_ seedlings to salt stress by scavenging ROS. The biosynthesis of AsA in plants is mainly accomplished through the D-mannose/L-galactose pathway. The key enzymes involved in this pathway include GMP, GME, GGP, L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH), and GLDH [32]. The GMP from _D. officinal_ is involved in the biosynthesis of mannan. The content of mannose in the _355:DoGMP_ transgenic _A. thaliana_ plants is higher than that in the wild type, thereby enhancing the tolerance of _A. thaliana_ plants to salt stress [33]. The relative expression level of some _CELLULOSE SYNTHASELIKE A_ (_CSLA_) genes which are involved in the synthesis of mannan in _D. officinal_ is upregulated (Figure 6B), suggesting that the content of mannan in the leaves of _D. officinal_ may increase under salt stress. The accumulation of AsA in High-Pigment-1 tomato is positively correlated with the expression level of _GMP_ and _GME_, but negatively correlated with the expression level of _GLDH_[31]. In this study, genes such as _GMP_, _GME_, _GLDH_, and _CSLA_ were likely to be transcriptionally regulated, modifying the final concentration of AsA in _D. officinal_ leaves under salt stress, so that the AsA content did not increase significantly under salt stress. The overproduction of ROS under salt stress triggers lipid peroxidation, harms photosynthetic pigments, and inhibits the photosynthetic rate. The AsA-GSH cycle can alleviate oxidative damage by scavenging excess ROS under abiotic stress [34]. The key enzymes involved in the AsA-GSH cycle in plants include ascorbate peroxidase (APX), glutathione reductase (GR), glutathione _S_-transferase (GST), and glutathione peroxidase (GPX). Overexpression of _A. thaliana_ APX in tobacco (_Nicotiana tabacum_) chloroplasts reduces the toxicity of H2O2, thus enhancing tolerance to salt stress [35]. Tobacco plants overexpressing soybean (_Glycine soja_) _GST_ show a higher tolerance at the seedling stage than wild-type plants to salt [36]. Overexpression of _Tritcium aestivum GPX_ confers strong tolerance to salt in _A. thaliana_[37].