## 2 Results  
### NR-Associated NO Is Involved in Enhancing Seed Germination under Salt Stress  
Salt stress was demonstrated to significantly inhibit seed germination in many species, such as _Arabidopsis_[43], soybean [12], cotton [44], and _Stylosanthes humtilis_[45]. In rice, seed germination was also significantly suppressed by salt stress in a concentration-dependent manner (Figure 1a), in which 100 mM sodium chloride (NaCl) was selected for the further experiments. Similar to other studies, application of SNP, a NO donor, has promoted seed germination of rice under salt stress (Figure 1b). Interestingly, the optimal concentration of SNP to increase seed germination rate under salt stress was 100 \(\upmu\)M. In contrast, application of c-PTIO, a NO scavenger, has aggravated the inhibition effect on seed germination in a concentration-dependent manner, too (Figure 1c). To further reveal the function of NO in improving salt tolerance during seed germination, a triple mutant of three nitrate reductase encoding genes, _OsNR1_, _OsNR2_ and _OsNR3_, was generated by CRISPR/Cas9 (Figure S1). Seedlings of two independent triple mutant lines, H5 and H10, were less vigorous than WT after 10 days of hydroponic culture  
Figure 1: Nitric oxide promotes seed germination under salt stress. (**a**) Seed germination under different concentrations of salt solution; (**b**) Seed germination rate at 60 h under CK (water) and 100 mM NaCl plus different concentrations of SNP (0, 50, 100, 150 and 200 \(\upmu\)M); (**c**) Seed germination rate at 60 h under CK and 100 mM NaCl plus different concentrations of cPTIO (0, 100, 200 and 300 \(\upmu\)M). The data are the means of three independent replications (\(n\) = 50) \(\pm\) SD. The asterisk (** and **) indicates a significant difference at the \(p\) < 0.05 and \(p\) < 0.01 level, respectively, and ns means not significant, by Student’s \(t\)-test analysis. (Figure 2a). However, the phenotype of triple mutants was similar to WT plant at t  
Figure 2: NR-associated NO is involved in enhancing seed germination under salt stress. (**a**) Phenotype of triple mutants, H5 and H10, at 7d-old hydroponic cultured seedlings using Kimura B nutrient solution; (**b**) Phenotype of triple mutants, H5 and H10, at 7d-old hydroponic cultured seedlings using ammonia-deprived nutrient solution; (**c**) NR activity in leaf and root of 14d\(-\)old seedlings; (**d**) NR activity in seed germinated under salt stress at 6 h and 12 h. (**e**,**f**) Seed germination of H5 and H10 under CK and 100 uM SNP; (**g**,**h**) Seed germination of H5 and H10 under 100 mM NaCl and 100 mM NaCl plus 1 mM NaNO\({}_{3}\). The data are the means of three independent replications (\(n=50\)) \(\pm\) SD. The asterisk (* and **) indicates a significant difference at the \(p<0.05\) and \(p<0.01\) level, respectively, and ns means not significant, by Student’s \(t\)-test analysis. NR-Associated NO Increased Salt Tolerance by Promoting Expression of ABA Catabolism Genes during Seed Germination under Salt Stress  
In our previous study, we found that NO was involved in seed dormancy breaking by inducing the expression of _CYP707A2_ gene, a key gene for ABA catabolism in _Arabidopsis_[30]. Furthermore, it was reported that ABA biosynthesis was induced by salt stress which thus inhibited seed germination of soybean and _Arabidopsis_[12; 43]. Here in rice, we also found that ABA content in seeds under salt stress was higher than that under CK condition at both 6 h and 12 h after imbibition (Figure 3a). Application of fluridone, an ABA biosynthesis inhibitor, can partially rescued seed germination under salt stress (Figure 3b). Then abundances of two key ABA biosynthesis gene, _OsNCED3_ and _OsNCED5_, were examined, results shown that _OsNCED3_ was significantly induced by salt stress in WT plant at 6 h after imbibition, while _OsNCED5_ displayed a similar expression trend with _OsNCED3_ but was only slightly induced by salt (Figure 3c).