## 2 Results
### NR-Associated NO Is Involved in Enhancing Seed Germination under Salt Stress
These results suggested that ABA biosynthesis were also induced by salt stress which is in agreement with previous studies. ABA level in plant is controlled by the antagonism between biosynthesis and catabolism [11]. To reveal the function of NR-associated NO on ABA catabolism during seed germination under salt stress, the expression of ABA catabolism genes was examined in WT and triple mutant seeds. Results shown that _OsABA8 ox1_ was induced by salt stress at 6 h after imbibition (Figure 4a). Interestingly, the expression of _OsABA8 ox1_ was down-regulated in both triple mutants under salt stress condition when compared with WT plant (Figure 4a), which was similar with the NR enzyme activity in WT and triple mutant seed under CK and salt conditions (Figure 2d), suggesting that _OsABA8 ox1_ might be regulated by NO during seed germination in response to salt stress. Expressions of _OsABA8 ox2_ and _OsABA8 ox3_ were not changed by salt stress in both WT and triple mutants (Figure 4b,c). ABA content was higher in both triple mutants than WT seed under CK condition. Under salt stress, ABA content was significantly increased in WT seed and triple mutants at both 6 h and 12 h after imbibition (Figure 4d). These results suggest that NR associated NO plays a key role in inducing the expression of ABA catabolism genes during seed germination under salt stress. Figure 3: Salt stress increased ABA biosynthesis in both WT and triple mutants during seed germination. (**a**) ABA content of WT seed germinated under water and 100 mM NaCl; (**b**) Seed germination under water and 100 mM NaCl plus different concentrations of fluridone (0, 10, 30 and 50 \(\upmu\)M); (**c**) Expressions of _OsNCED3_ and _OsNCED5_ in WT germinated under water and 100 mM NaCl solution. The data are the means of three independent replications (\(n=20\)) \(\pm\) SD. The asterisk (* and **) indicates a significant difference at the \(p<0.05\) and \(p<0.01\) level, and ns means not significant, respectively, by Student’s \(t\)-test analysis. OsNLP2 Was Induced by Salt Stress to Upresulate Expression of OsNRs and Therefore Enhance the NR Activity  
Nitrate was well reported to promote seed dormancy breaking and germination in different species [35]. However, whether and how nitrate is involved in salt response during seed germination is still obscure. In this study, we found that application of 1 mM nitrate could slightly enhance germination rate of _Nipponbare_ (WT) seed. Whereas under salt stress, exogenous nitrate has significantly promoted seed germination (Figure 5a). Then we examined the expression of _OsNLP_ gene family by qPCR and found that _OsNLP2_, the rice orthologole of _AtNLP8_ in _Arabidopsis_, was significantly induced by salt stress at 6 h after imbibition (Figure 5b). Similar to _OsABA8 ox1_ which is a possible target for NLP2, _OsNR1_ and _OsNR2_ were also induced by salt stress at 12 h (Figure 5c). Application of nitrate during seed germination under salt stress has significantly increased the expression of _OsNR1_ and _OsNR2_, though the expression of _OsNLP2_ was not further promoted by nitrate (Figure 5d). Since NLP transcript factors have been well reported to activate the expression of _OsNRs_ and _OsNRs_ genes, we thus speculated that NLP2-NR module might be crucial in mediating nitrate-promoted seed germination under salt stress. Figure 4: NR-associated NO increased expression of ABA catabolism genes during seed germination under salt stress. (**a**–**c**) Expressions of _OsABA8 ox1_, _OsABA8 ox2_ and _OsABA8 ox3_ in WT, H5 and H10 germinated under water and 100 mM NaCl solution; (**d**) ABA content of WT and triple mutant seeds germinated under water. The data are the means of three independent replications (\(n\) = 20) \(\pm\) SD. The asterisk (**c** and **d**) 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. Different letters a, b, c and others indicate significant differences, \(p\) < 0.05, by [PERSON]’s test analysis.