The chemical background of NO becomes complicated when it is applied to biological systems [72]. The main reason behind this is the rapid reaction of NO with oxygen to form different nitrogen oxides; on the other side, several factors such as the concentration and system redox states of NO, along with the concentrations of its target molecules and metals, regulate its stabilization [73]. Delledonne [74] demonstrated some contradictory effects of HMs on the internal concentration of NO in plants as a result of changes in both the concentration and redox state of NO. In addition to such controversy, some other factors have also been identified and found responsible, such as difficulties in detecting NO, and, moreover, in measuring it [75]. However, NO synthesis in plants by purified mitochondria and peroxisomes and NR have potentially been measured via many different types of methods. Examples include chemiluminescence, DAF-fluorescence, fluorescence imaging, electron paramagnetic resonance spectroscopy (EPR), oxyhemoglobin/methemoglobin, laser photo-acoustics, NO electrodes, and mass spectrometry [76,77,78,79,80].

The content of NO in different parts of different plants may either increase or decrease depending upon the type of HM, as represented in Table 1.

The levels of NO have mainly been studied under Cd stress. For instance, wheat roots were kept for 5 days [91] or 3 h [83] under Cd stress. Both cases showed a rise in the NO content. However, in the case of rice, both roots and shoots kept for 7 days [92] and only rice roots kept for 24 h [72] under Cd stress showed a decline in the NO content. Studies with Cd-stressed Pisum sativum roots exposed for 7 days showed an increased level of NO [85], while the same set kept for 15 days showed a decrease in the NO content [94], but Cd-stressed leaves of Pisum sativum kept for 14 days indicated a decreased amount of NO [95,96].

Both a cell suspension of Glycine max [86] and Nicotiana tabacum [90] exposed to Cd stress for 72 h and 12 h, respectively, displayed a rise in the NO content. Roots of Medicago truncatula were kept two times (48 h) longer than roots of Hordeum vulgare (24 h) under Cd-stressed conditions. The NO content increased in the roots of H. vulgare [88], while M. truncatula roots exhibited a decrease in NO production. Both a cell suspension of A. thaliana kept under Cd for 72 h [82] and the same material kept under Fe for 30 min [84] displayed an increased amount of NO. Seedlings of A. thaliana also exhibited a rise in NO levels when incubated for 14 days under exposure to Pb [77]. Interestingly, a drop in NO content was observed in the roots of A. thaliana kept under Al stress for an hour [81]. However, leaves of A. thaliana kept under Cd exposure for 96 h displayed a rise in NO levels [35,83].

Bartha et al. [85] used the roots of Pisum sativum and Brassica juncea and kept them under Cu stress for 7 days. After the incubation period of 7 days, both sets showed an increase in the NO content. Similar results were found with the same plant root specimens under Zn stress [85]. For experimental purposes, [98] kept the roots of Solanum nigrum under two different stressed conditions; the first set contained Zn, while the second set included Fe along with Zn, and these two sets were incubated for 0 to 10 days. The result showed an increase in the NO content for 2–3 days, and then it started to decline.

As many different factors, such as the concentration and treatment time of HMs, variety of plant species, size and shape of the plant at the time of HM treatment, etc., are thought to be associated with changes in endogenous NO content in plants, there has been much debate among researchers about the actual cause of such change. One of the possible reasons for the decrease in the endogenous NO content, mentioned by [99], is that calcium deficiency has been observed in plant leaves under Cd stress, which disturbed NOS-like enzyme activity; as a result of this, the endogenous NO content is reduced. An increase in NR enzymatic activity [100,101] and the genotypes of plants [102,103] have been referred to as potential factors that can cause an increase in NO content in Cd-stressed plants. Apart from that, as NO can readily react with oxygen and form nitrogen oxides, the balance between the binding state and intracellular redox state of some specific smaller molecules to NO can also be considered a responsible factor for the increase or decrease in the endogenous NO content in plants [102,103].

Over the past few decades, rapid industrialization has contaminated natural resources to some great extent [104]. As a result of such industrial development, HM toxicity [105] has adversely affected plants, animals, microorganisms [106], and, all in all, the entire ecosystem [104].

As a multipurpose gaseous signaling molecule [36,107], NO makes a powerful contribution in inducing plants to stand against the toxic attack of HMs  through both exogenous and endogenous application [107].

A very well-suited example includes exogenous application of NO in both rice and Vigna radiata L. seedlings under As stress conditions, in which NO was able to ameliorate the toxic effects of heavy metal As by minimizing the levels of ROS and malondialdehyde (MDA). A similar kind of result of overcoming HM toxicity in rice by NO application was reported in another study against Cu stress [108]. In Typha angustifolia, NO demonstrated remarkable improvement in plant growth and development and also in the total biomass yield by suppressing Cd stress [106].

Experiments were performed on rice seedlings (Oryza sativa L.) under Cd and As stress to investigate whether NO could ameliorate both these HMs’ toxicity or not [71]. Their studies revealed that under Cd stress, the endogenous concentration of NO can diminish alterations in the root system, but it is unable to suppress the majority of damage caused by As. Xiong et al. [72] reported that NO enhances the pectin and hemicelluloses content in the cell wall of the root system of rice, which helps to stop the accumulation of Cd in the leaves of rice seedlings. An indirect contribution of NO was observed to alleviate Cd toxicity with the help of Bacillus amyloliquefaciens SAY09 acting downstream of auxin by activating the auxin-mediated signaling pathway [107]. Recent studies have reported the ability of NO in decreasing AsIII toxicity by the modulation of jasmonic acid (JA) biosynthesis [88].

Emamverdian et al. [110] proved through their experiments that SNP (sodium nitroprusside, a strong nitric oxide donor) can reduce the accumulation of two specific HMs, Pb and Cd, in the root and shoot system in plants. In addition to that, under HMs like Pb and Cd-stressed conditions, the NO donor showed remarkable contributions in many parameters, including increases in the protein, non-protein, and total thiol contents; protection of the plasma membrane and cell-developing antioxidant-enzyme activities in bamboo plants; elongation of the shoot length; and many more [110]. The application of SNP has shown some incredible results in reducing the effect of Cd-generated ROS production [61] and regulating the metabolic state of antioxidation in some crops, such as mustard [116], wheat [117], rice [104], and peanut [118]. SNP was added to Cd-stressed rice plants at low concentrations, and the result of this experiment showed stimulation of the Cd tolerance of rice by increasing the pectin and hemicellulose contents in the root cell wall [72]. In this context, Correa-Aragunde et al. [118] performed experiments on tomato plants by applying both low and high concentrations of NO individually, in which enhancement of cellulose synthesis was observed in tomato roots, while, on the other hand, application of NO donor in a higher concentration showed reverse results. According to Lombardo et al. [64], NO plays a very significant role as a positive regulator in root hair development. Studies have shown that the exogenous application of NO helps to decrease Al accumulation in the root apical zones of rye and wheat seedlings [104].