## 3 Discussion
### NR-Associated NO Plays a Key Role in Regulating Seed Germination and Salt Tolerance  
In plants, there are several NO origins which can be divided into enzymatic pathway and non-enzymatic pathway [20]. Many studies have demonstrated that NR-dependent pathway was the major source for NO production and plays a key role in regulating plant NO homeostasis [54; 55]. Both pharmacological and genetical evidence have supported that NR is crucial for NO production in plants [49]. Application of tungstate, sodium azide and potassium cyanide will significantly reduce the NO production in plant [56; 57]. While mutation of nitrate reductase genes and _AINOS1_ simultaneously has led to a reduced NO production, inhibition of seed germination and reduced plant size in _Arabidopsis_. Application of SNP was able to significantly, but not fully, promote seed germination of this triple mutant [19]. Here in this study, we found that triple mutant of three nitrate reductase genes in rice displayed a delayed germination phenotype which was also partially rescued by exogenous NO (Figure 2e,f). Our results further supported that NR-associated NO production is crucial in regulating seed germination. In contrast with NO, the function of NR in salt tolerance has not yet been well demonstrated. Nevertheless, it was reported that NR-dependent NO production was important in response to salt stress in cucumber root when subjected to 50 mM sodium chloride [42] and in soybean during seed germination under 50 mM sodium chloride [41]. However, these results were all based on pharmacological experiment by using sodium tungstate which might have other side effect on plant. Interestingly, deficiency of NO production in the triple mutant of _Arabidopsis_ displayed an increased dehydration tolerance by efficiently regulating stomata close when compared with WT plant [19], suggesting that NR might play a negative role in response to water stress. In the present study, we found that NR activity was increased by salt stress in the early stage of seed germination (Figure 2d). In addition, the enhanced NR enzyme activity by salinity was blocked in the triple mutants, H5 and H10 (Figure 2d), resulting in higher sensitivity to salt stress during seed germination than seed of WT plant (Figure 2g,h). Application of SNP to these two triple mutants has significantly reduced the sensitivity and thus promoted seed germination of H5 and H10 under salt stress (Figure 2g,h). This genetic evidence strongly indicates that NR plays a key role in promoting salt tolerance during seed germination of rice by producing NO. Phytohormone ABA plays a crucial role in salt response by acting as a central integrator that links the complicated developmental processes and salt stress [9]. ABA is significantly induced by salt stress to deal with osmotic stress and oxidative stress [10]. However, the content of ABA should be carefully controlled during seed germination under salt stress because accumulation of ABA will suppress seed germination and even aggravate oxidative stress via ABI4-RbohD pathway [43]. Indeed, ABA content in salt treated seed at 6 h after imbibition was higher than that in CK condition due to the up-regulation of _OsNCD_3 gene by salt (Figure 3a,c). In addition, application of ABA biosynthesis inhibitor has mitigated the suppression effect of salinity on seed germination (Figure 3b), indicating that ABA biosynthesis was induced by salt stress. Interestingly, the expression of _OsABAB8 ox1_ gene was also up regulated during seed germination under salt stress in WT plant but not triple mutant (Figure 4a), leading to a higher level of ABA in seed of H10 (Figure 4d). NO production during the early stage of seed germination has induced the expression of _CYP707A2_, a key ABA catabolism gene in _Arabidopsis_, which thereby promoted seed germination [30]. Furthermore, ABA content in plants is controlled by an antagonism between biosynthesis and catabolism under normal condition and abiotic stresses [11].