## 3 Discussion
### OsNLP2 Was Involved in Salt Defense by Up-Regulating Expression of Both OsNRT1 and OsABA8 ox1 Genes  
NIN-like protein (NLP) belongs to the transcript factor family which act as master regulator in nitrate response by binding to nitrate responsive cis-element (NRE) in many nitrogen responding genes in _Arabidopsis_[58; 59]. In rice, 6 _NLP_ genes has been identified based on in silico analysis [21]. _OsNLP1_, _OsNLP3_ and _OsNLP4_ were reported to play key role in improving yield and nitrogen use efficiency. Over expression of these genes all displayed higher yield per plant by increasing tiller number and grain number per plant. What is more, three _OsNLP_s were all found to bind the promoter of _OsNR1_, _OsNR2_ and _OsNiR_ which thereby regulate the expression of these genes [37; 38; 46]. Their results strongly indicated that _OsNLP_s might be involved in regulating NO production via NR-NiR pathway. Although functions of _OsNLP_s have been well illustrated in nitrogen response, whether their play a role in stress defense has not yet been reported. In this study, we found salt stress has induced the expression of _OsNLP2_ gene at early stage of seed germination (Figure 5b), accompanied by up-regulation of _OsNR1_/2 (Figure 5c) and increased NR activity (Figure 2d). Similar with _OsNLP1_, _OsNLP3_ and _OsNLP4_ genes, _OsNLP2_ was also found to regulate the expression of _OsNR1_ and _OsNR2_ in our transient trans-activation expression assay (Figure 8a,b). Together, these results further proved that _OsNLP2_ is involved in NO production via regulating expression of _OsNR_s in response to salt stress.  
Nitrate has been well known to promote seed dormancy breaking and germination [60]. _AtNLP8_, as a nitrate responsive gene, was activated by application of nitrate and thus increased the expression of _CYP707A2_ gene during seed germination in Arabidopsis [35]. In addition, _CYP707A2_ plays a key role in regulating seed germination and is activated by NO during seed germination in _Arabidopsis_[30; 61], implying that nitrate-activated NLP8 might reduce ABA level by directly binding to promoter of _CYP707A2_ and indirectly inducing the expression of _CYP707A2_ via enhancing NO production during seed germination in Arabidopsis because the expression of _AtNIA2_, orthologique of _OsNR2_, was induced by nitrate and blocked in _nlp8_ mutant. In the present study, it is demonstrated that application of nitrate has significantly alleviated the inhibition effect of salinity on seed germination (Figure 5a). Expression of _OsNR1_ and _OsNR2_ was further induced by nitrate during seed germination under salt stress (Figure 5d). However, the promoting effect of nitrate on seed germination was blocked in triple mutants under both CK and salt stress conditions (Figure 6), suggesting nitrate-promoted seed germination was partially mediated by NLP2 NR module-dependent NO production. In addition, expression of _OsABA8 ox1_ was induced by salt stress in WT plant, which is partially reduced in both H5 and H10 under CK or salinity conditions (Figure 4a). Application of nitrate to mutants of ABA catabolism genes and its corresponding wide type plant, Zhong Hua 11 (ZH11), shown that seed of _abu8 ox1_ was insensitive to nitrate (Figure 7a,c), while _abu8 ox2_ was partially promoted by nitrate during seed germination (Figure 7b-d). Luciferase assay further revealed that OsNLP2 activated the expression of _OsABA8 ox1_ gene in response to nitrate application during seed germination under salt stress (Figure 8c).