The present study unravels origin of nitric oxide (NO) and the interaction between 24-Epibrassinolide (EBL) and nitrate reductase (NR) for NO production in Indian mustard (Brassica juncea L.) under salinity stress. Two independent experiments were performed to check whether (i) Nitrate reductase or Nitric oxide synthase takes part in the biosynthesis of endogenous NO and (ii) EBL has any regulatory effect on NR-dependent NO biosynthesis in the alleviation of salinity stress. Results revealed that NR-inhibitor tungstate significantly (P ≤ 0.05) decreased the NR activity and endogenous NO content, while NOS inhibitor l-NAME did not influence NO biosynthesis and plant growth. Under salinity stress, inhibition in NR activity decreased the activities of antioxidant enzymes, increased H2O2, MDA, protein carbonyl content and caused DNA damage, implying that antioxidant defense might be related to NO signal. EBL supplementation enhanced the NR activity but did not influence NOS activity, suggesting that NR was involved in endogenous NO production. EBL supplementation alleviated the inhibitory effects of salinity stress and improved the plant growth by enhancing nutrients, photosynthetic pigments, compatible osmolytes, and performance of AsA-GSH cycle. It also decreased the superoxide ion accumulation, leaf epidermal damages, cell death, DNA damage, and ABA content. Comet assay revealed significant (P ≤ 0.05) enhancement in tail length and olive tail moment, while flow cytometry did not showed any significant (P ≤ 0.05) changes in genome size and ploidy level under salinity stress. Moreover, EBL supplementation increased the G6PDH activity and S-nitrosothiol content which further boosted the antioxidant responses under salinity stress. Taken together, these results suggested that NO production in mustard occurred in NR-dependent manner and EBL in association with endogenous NO activates the antioxidant system to counter salinity stress.

中文翻译：

---

盐胁迫下芥菜油菜中外源24种油菜素内酯与内源性NO的相互作用：NR依赖型NO生物合成的证据。

本研究揭示了盐分胁迫下印度芥菜（芥菜）中一氧化氮（NO）的起源以及24-表油菜素内酯（EBL）和硝酸还原酶（NR）之间的相互作用。进行了两个独立的实验，以检查（i）硝酸还原酶或一氧化氮合酶是否参与内源性NO的生物合成，以及（ii）EBL对缓解NR依赖性NO的生物合成是否具有减轻盐分胁迫的调节作用。结果表明，NR抑制剂钨酸盐显着（P≤0.05）降低了NR活性和内源性NO含量，而NOS抑制剂l-NAME不影响NO的生物合成和植物生长。在盐分胁迫下，抑制NR活性会降低抗氧化酶的活性，增加H2O2，MDA，蛋白质羰基含量并造成DNA损伤，暗示抗氧化防御可能与NO信号有关。补充EBL可增强NR活性，但不影响NOS活性，表明NR参与内源性NO产生。EBL补充剂通过增强营养素，光合色素，相容的渗透压剂和AsA-GSH循环的性能，减轻了盐分胁迫的抑制作用并改善了植物的生长。它还减少了超氧化物离子的积累，叶片表皮损伤，细胞死亡，DNA损伤和ABA含量。彗星试验显示，盐度胁迫下尾巴长度和橄榄色尾矩显着增强（P≤0.05），而流式细胞仪未发现基因组大小和倍性水平发生显着变化（P≤0.05）。此外，EBL补充增加了G6PDH活性和S-亚硝基硫醇含量，进一步增强了盐分胁迫下的抗氧化反应。综上所述，这些结果表明芥菜中的NO产生是以NR依赖的方式发生的，而EBL与内源性NO结合会激活抗氧化剂系统以抵抗盐分胁迫。