1. Introduction

The loss of millions of hectares of arable lands to various stresses every year continues to impose a major threat to agriculture. Almost 90% of all arable lands have been exposed to major environmental stresses, such as salinity, drought, extreme temperature, heavy metals, and ultraviolet radiation ^[1]. Of these, water deficit caused by drought is amongst the main threats to crop growth and production worldwide. In the US alone, the combined effect of drought and increased temperature has caused a total loss of about USD555 billion for the past 10 years (2010–2019) in agricultural production ^[2]. The effect of drought is even more devastating in developing countries whose economies largely rely on their agricultural output. According to the Food and Agriculture Organization of the United Nations (FAO), 30% of agricultural losses (USD ~29 billion) due to drought was recorded in developing countries in the period of 2005–2015 ^[3].

Drought conditions cause a disruption to the cellular redox homeostasis in plants, leading to oxidative stress and cell injury ^[4]. Plants respond and adapt to drought stress by changing their morphology, biochemistry, physiology, and molecular mechanisms ^[5]. One of the major signaling molecules present in the cascade of this stress adaptation response is nitric oxide (NO), a water- and lipid-soluble free radical and a gaseous, redox-related signaling molecule that is rapidly produced by multiple hormonal and environmental stimuli ^[6]. NO regulates and maintains the level of reactive oxygen species (ROS) by inducing transcriptional changes of various targets involved in plant defense and cell death, translocation, signal transduction, and ROS metabolism ^[7].

Some studies have supported the roles of NO in drought adaptation, but little is known about this important molecular amendment in modulating signaling in abiotic stresses. Studies regarding the origin and production of NO during water deficit, its sensing, and transduction as well as its physiological and molecular processes for the amelioration of drought stress remain scarce. Continued efforts in examining the roles of NO during plant adaptation to drought stress and its detection and measurement methods are, therefore, crucial for facilitating breeding and crop improvement programs to counter the challenges faced by the agricultural industry.

2. Biosynthesis of NO in Plants

NO biosynthesis has been well characterized in mammals. It is mainly synthesized through NO synthase (NOS) activity . In plants, NO can be biosynthesized via reductive and oxidative routes ^[8]. The most thoroughly researched NO biosynthetic pathway in plants is the reduction of nitrite, either enzymatically or nonenzymatically. Enzymes that have been demonstrated to catalyze the generation of NO from nitrites are nitrate reductase (NR) ^[9], the membrane-bound nitrite NO reductase (NiNOR) ^[10], and peroxisomal xanthine oxidoreductase (XOR) ^[11]; mitochondrial electron transfer chain-dependent enzymatic nitrite reduction has also been reported to be capable of this ^[12]. In contrast, nonenzymatic reduction has been reported to occur in the apoplast of barley aleurone layers under acidic environments with the presence of high concentrations of nitrate (NO₂⁻) ^[13]. The oxidative route of NO production relies on the oxidation of aminated molecules, such as L-arginine(L-Arg) by the L-Arg-dependent NOS-like enzyme ^[14], polyamines ^[15], and hydroxylamine ^[16].

3. The involvement of NO in Drought Tolerance

The role of NO in mitigating drought stress effects has been observed in several plant species, including grains, legumes, fruit trees, medicinal plants, and vegetables. The production of NO depends on the drought stress level, exposure duration, and plant growth stages. For instance, the accumulation of NO in Cucumis sativus increased from 75-fold within the first 10 h of drought exposure to 190-fold after 17 h of drought exposure compared to well-watered plants

^[17]. Different types of nitrogen nutrition supplied to plants have been reported to influence the rapidness of endogenous NO production. Cao et al. ^[11] reported that rice supplemented with ammonium (NH₄⁺) induced endogenous NO production after 3 h of drought treatment, whereas those supplemented with NO₃⁻ induced NO production after 6 h of drought treatment. Their findings indicate that NH₄⁺ is more effective than NO₃⁻ in alleviating water stress, probably due to the early NO burst which might be triggered by NOS-like enzymes in roots.

Exogenous application of NO-induced stomatal closure in Tradescantia sp., Salpichroa organifolia, and Vicia faba ^[18

]

. The saturated net carbon dioxide assimilation rate, stomatal conductance, substomatal carbon dioxide concentration, and transpiration rate were also increased after NO treatment ^[19]. By applying the NO scavenger 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), stomatal closure was inhibited, supporting the notion that NO is induced endogenously and most likely could act as a signaling element to facilitate stomatal closure. In addition, NO may promote the formation of guard cells and influence the distribution of stomata in the abaxial leaf ^[20].

4. Detection of NO: Challenges

The quantification of NO is technically challenging due to its unique chemical properties. NO is produced in subcellular compartments in a broad range of concentrations, from pico- to micro-molar. It diffuses freely across membranes and has a short half-life (seconds) in biological systems

^[21]

. In addition, NO signaling displays complicated temporal and spatial arrangements which further impedes its detection

^[21]

. Due to its ubiquity and involvement in various intrasignaling processes, the analytical methods used for its detection and analysis should suit the biological model and take into account the distinct features of NO production and functions. For example, NO generation in barley infected by Blumeria graminis f. sp. hordei (powdery mildew) was reported by a subtle change in the induction of the epidermal or the formation of cell wall papillae, which can be very difficult to detect using only a gas-based NO detection system

^[22]

. Due to these difficulties, questions have been raised regarding the roles of NO in certain biological processes and the specificity of the detection methods used.

Fluorescent probes have been widely used to detect cellular NO due to their excellent sensitivity and selectivity. Most fluorescent probes, however, not only bind to NO but also bind to its oxidation by-products, other reactive secondary species or divalent metals, such as dinitrogen trioxide (N

₂

O

₃

), NO

₃⁻

, peroxidases, H

₂

O

₂

, ascorbic acid, dehydroascorbic acid, and calcium chloride

^[23]

. One such example is diaminofluorescein (DAF) dyes (DAF-2 and DAF-2DA). The main drawback of the DAF dye method is its tendency to react with other ROS, such as peroxidases and H

₂

O

₂

^[23]

. Recently, an electrochemically based method using a homemade platinum- or iridium-based electrochemical microsensor has been reported to provide real-time detection of NO production in plant cell suspension cultures

^[21]

. In addition, another electrochemical sensing microbundle that can simultaneously measure NO, H

₂

O

₂

, and pH under drought stress at various developmental stages was invented

^[24]

.

Chemiluminescence is a chemical assay used to detect NO based on NO reaction with ozone. Upon reacting with ozone, NO forms nitrogen dioxide at an excited state. As it decays into the ground state, it emits a photon that can be detected by a photomultiplier tube

^[25]

. Since the photon emitted is proportional to the reacting NO, this allows a reliable estimation of NO. This method holds advantages over other NO detection methods due to its sensitivity (detection in the pmole range) and ability to directly measure NO in both aqueous and gaseous states

^[26]

. The disadvantages of the chemiluminescence method include reading errors that could be introduced due to the sensitivity of the photomultiplier or changes in the light generated by the secondary source of light. Another disadvantage is that chemiluminescence only measures gaseous NO and no other forms of nitrogen or reactive nitrogen intermediates

^[27]

. Thus, the considerable levels of NO that are oxidized in living tissues cannot be easily detected using this method.

“Spin-trapping” is a commonly used method involving the detection of a transient, reactive free radical using a high concentration of diamagnetic spin trap. This approach is known as electron paramagnetic resonance (EPR) spectroscopy (also known as electron spin resonance, ESR, or electron magnetic resonance, EMR)

^[28]

. Since NO and superoxide are diatomic free radicals, their presence could be detected using this method. NO can be effectively measured in the gas phase using EPR; however, its signal is less sensitive in the liquid phase

^[29]

. Since the steady-state level of NO is below the detectable limit of EPR, NO needs to be trapped in a more stable form to allow its proper detection. This is achieved by adding a reactive free radical to the double bond of a diamagnetic compound known as “spin trap,” forming a more persistent and stable secondary free radical (radical adduct)

^[30]

. An example is nitrone 5,5-dimethyl-1-pyrroline N-oxide (DMPO) which is used to trap free radicals, such as superoxide and hydroxyl, to provide the radical adducts with a distinctive spectrum. Unlike superoxides, which are highly stable and allow direct measurement, NO depends on reaction with other free radicals or its ability to coordinate with iron ions for a more reliable measurement

^[31]

. 

Developing efficient and sensitive methods of detection is clearly indispensable for the accurate detection and quantification of NO. Mur et al.

^[22]

suggested the use of more than one technique to detect NO. In a study by Bright et al.

^[32]

, a combination of the DAF dye technique and EPR spectroscopy was applied to measure NO production when hydrating pollen. Besson-Bard et al.

^[33]

combined the NO probe DAF-2 and an electrochemical method to detect the role of cryptogein, a fungal elicitor, in increasing NO production under biotic stress in tobacco. A similar approach was used to measure NO production in tobacco suspension cells

^[27]

. The recent advances in quantum cascade laser-based spectroscopy have offered new opportunities to measure gaseous NO based on the absorption of laser light by the NO molecules and provide online planta measurements of the dynamics of NO production. Despite its sensitivity, this detection technique also has its flaws, including unintended interferences with another species, unreliable sensitivity, and indirect measurement taken from the secondary NO species

^[34]

.