Background

Nitric oxide (NO) is emerging as a key regulator of diverse plant cellular processes like regulating synthesis of the cell wall (Correa-Aragunde et al., 2008; Xiong et al., 2009; Ye et al., 2015), ROS metabolism in plants (Delledonne et al., 2001), gene expression and regulation (Bogdan et al., 2000), programmed cell death (de Pinto et al., 2002), maturation and senescence (Yaacov et al., 1998). NO exerts a crucial role in protecting plants against various abiotic stresses (Hung et al., 2002). NO could significantly enhance antioxidative capacity by increasing the activities of catalase (CAT), ascorbate peroxidase (APX) and accumulating proline during wheat seed germination under osmotic stress (Zhang et al., 2003). A major route for the transfer of NO bioactivity is S-nitrosylation, the addition of a NO moiety to a protein cysteine thiol forming an S-nitrosothiol (SNO). Total cellular levels of protein S-nitrosylation are controlled predominantly by S-nitrosoglutathione reductase 1 (GSNOR1) which turns over the natural NO donor, S-nitrosoglutathione (GSNO). In the absence of GSNOR1 function, GSNO accumulates, leading to dysregulation of total cellular S-nitrosylation (Yun et al., 2016)

Nitric oxide (NO) production in land plants classically involves two main routes: first, a reductive pathway involving both enzymatic and non-enzymatic reduction of nitrite into NO (Gupta et al., 2011); second, an oxidative pathway requiring a putative nitric oxide synthase (NOS)-like enzyme. Role of NR in NO production was suspected by low or no NR activity mutants which show no measurable NO. Later nia1/nia2 double mutants of Arabidopsis confirmed the role of NR in reduction of NO₂^- to NO in NADH dependent manner under both in vitro (Yamasaki et al., 1999) and in vivo (Vanin et al., 2004) condition. The possibility that NOS could catalyze NO synthesis in plants has also been a main controversial issue. Experimental evidence further increased suspicion about the existence of a plant NOS-like enzyme. It was reported that the L-citrulline based assay commonly used to measure a NOS activity in plant extracts is prone to artefacts (Tischner et al., 2007).

Several methods have been reported for nitric oxide assay in plants which includes gas chromatography and mass spectrometry (Neil et al., 2003; Conarth et al., 2004, Bethke et al., 2004), laser photo-acoustic spectroscopy (Lesham and Pinchasov, 2000), NO electrode (Yamasaki et al., 2001), electron paramagnetic resonance (EPR) (Sun et al., 2018) and a group of florescent dye indicators which are available in acetylated form for intracellular measurements like Diaminofluorescein-FM (DAF-FM) (Du et al., 2016). Fluorescent dye indicator and EPR both are highly specific for NO. EPR is limited by inability to detect low level NO production and insolubility of chelating agent. Use of fluorogenic probe DAF-FM is gaining more importance because of their simplicity, high sensitivity towards NO and are essentially independent of pH above pH 5.5. This probe is membrane-permeant and deacylated by intracellular esterases to 4-amino-5-methylamino-2 V, 7 V-difluorofluorescein. Presence of light leads to autoxidation of Diaminofluorescein-FM (DAF-FM) dye and simultaneous presence of NO and superoxide source (like xanthine + xanthine oxidase) decreases the fluorescence of Diaminofluorescein-FM (DAF-FM), resulting in underestimation of nitric oxide production (Balcerczyk et al., 2005). This limits the use of Diaminofluorescein-FM (DAF-FM) in stress-related study.

The activity of NOS can be measured by L-citrulline based assay (Tischner et al., 2007) and spectroscopic method (NADPH utilization method) (Gonzalez et al., 2012). Citrulline-based assay measures the formation of L-citrulline from L-arginine using ion exchange chromatography. The assay does not exactly quantify citrulline; any arginine derivative that does not bind to the cation exchange resin will give a signal and leads to false measurement and also involve radiolabelling which may be tedious to handle (Tischner et al., 2007). Spectrophotometric measurement of NOS activity has been widely regarded as a less expensive and sufficiently sensitive assay for routine laboratorial experiments.

Nitrate reductase activity can be measured by an in vitro or an easy to perform in vivo method (Nair and Abrol, 1973). We have used these protocols to study the effect of elevated CO₂ (EC) and nitrate supply on nitrogen metabolism in wheat seedlings (Adavi and Sathee, 2019). S-nitrosylation of NR by EC induced NO produced in plants supplied with high nitrate concentration decreases the enzyme activity (Cheng et al., 2015; Du et al., 2016).