**3. NO-phytohormone cross talk under heavy metals stress**

Heavy metals (HMs) are phytotoxic elements that can damage plant growth and metabolism at very low concentrations [46]. The involvement of plant hormones such as IAA, CK, and ET to alleviate HMs-induced toxicity is well reported [47–49]. Some recent studies suggest that NO acts in concert with signaling pathways of phytohormones to induce tolerance against excess elements [50, 51]. However, the exact nature of NO-hormone interactions still needs to be explored and is largely dependent on the species, the plant organ as well as concentration of metal and duration of stress [52].

Cadmium is one of the most widely distributed HM in agricultural soils [53]. Cd-induced increase in endogenous levels of NO is associated with its role as a bioactive molecule to quench ROS [54]. Alterations in hormonal homeostasis are potential signals that directly affect plant responses to Cd stress, including interplay between hormones and the whole plant signaling network, such as the ROS [55], MAPK [56], and NO signaling pathways [57]. Exposure to short-term Cd stress revealed an interrelation of ET with NO generation, polyamine metabolism, and MAPK cascades in young *Glycine max* seedlings [58]. It is well documented that exposure to HMs enhances the production of ET [59] due to increased 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) activity in metal stressed plants [60]. Cross talk between ET and stress signaling molecules like NO is important to understand the mechanisms of plant adaptation to HM-induced oxidative stress [61–63]. More recently, Thao et al. [51] suggested a possible link between NO and ET through MAPKs in plants exposed to HM stress. Accumulation of ET reduced NO levels and promoted the Cd-induced senescence processes in *Pisum sativum* [64]. Similarly, integration of ET, NO, PA, and MAPKs pathways improved tolerance in young *Glycine max* seedlings against short-term Cd stress [52]. The potential involvement of ETHYLENE INSENSITIVE2 (EIN2) in improving resistance against lead (Pb) stress has also been demonstrated in *Arabidopsis* [65]. Treatment of *Triticum aestivum* seedlings with Cd and PAs (spermine and putrescine) was found to induce NO generation in roots leading to root growth inhibition [66].

Experiments with *Arabidopsis* showed that Cd toxicity triggers NO accumulation, which in turn promotes the stabilization of AUX repressor protein IAA17 through suppression of AUX carriers PIN1/3/7 [67]. Xu et al. [68] found that exogenous NO supply improved the antioxidative capacity and reduced the degradation of AUX in roots of *Medicago truncatula* seedlings exposed to Cd stress. In another study, it was noted that NO acts downstream of AUX on modulating root architecture in *Arabidopsis seedlings* exposed to Cd stress [69]. Rodríguez-Serrano et al. [70] demonstrated that Cd toxicity strongly depressed the activity of NO-synthase dependent NO production in *Pisum sativum* seedlings; however, exogenous Ca supply ameliorated this effect due to enhanced JA and ET production. Interplay among NO, H2 O2 , and SA has also been reported in lupine seedlings [71] and ryegrass plants [72] under Cd stress. Application of putrescine (Put) and NO in combination was reported to alleviate Cd toxicity in *Vigna radiate* by triggering the activity of both enzymatic and nonenzymatic antioxidant machinery and a parallel increase in phytochelatin synthesis [73].

A positive interaction between NO and CK under water-limited environment was reported by Shao et al. [42]. Treatment of plants with CK plus NO scavenger (Hemoglobin) revealed that CK promoted NO signaling, probably mainly through a NR source in plants exposed to water stress conditions. CK interaction with NO signaling cascades regulated photosynthetic machinery and increased the adaptability to drought stress in *Zea mays* [42]. Contrasting reports indicate antagonistic interaction between NO and CK, for example, CK-induced reduction in NO levels promoted stomatal opening in dark grown *Vicia fabia* seedlings [43]. Presumably, CKs activate plasma membrane H1-ATPase through decreasing NO levels in guard cells, and then stimulate stomatal opening in darkness. Wilhelmova et al. [44] reported similar results in transgenic tobacco plants. Negative interaction between NO and CKs was evident during leaf development as increased NO production reduced CKs level in aging leaves. Evidence suggests that reaction of zeatin with peroxynitrite, a NO derivative, reduces

Heavy metals (HMs) are phytotoxic elements that can damage plant growth and metabolism at very low concentrations [46]. The involvement of plant hormones such as IAA, CK, and ET to alleviate HMs-induced toxicity is well reported [47–49]. Some recent studies suggest that NO acts in concert with signaling pathways of phytohormones to induce tolerance against excess elements [50, 51]. However, the exact nature of NO-hormone interactions still needs to be explored and is largely dependent on the species, the plant organ as well as concentration

Cadmium is one of the most widely distributed HM in agricultural soils [53]. Cd-induced increase in endogenous levels of NO is associated with its role as a bioactive molecule to quench ROS [54]. Alterations in hormonal homeostasis are potential signals that directly affect plant responses to Cd stress, including interplay between hormones and the whole plant signaling network, such as the ROS [55], MAPK [56], and NO signaling pathways [57]. Exposure to short-term Cd stress revealed an interrelation of ET with NO generation, polyamine metabolism, and MAPK cascades in young *Glycine max* seedlings [58]. It is well documented that exposure to HMs enhances the production of ET [59] due to increased 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) activity in metal stressed plants [60]. Cross talk between ET and stress signaling molecules like NO is important to understand the mechanisms of plant adaptation to HM-induced oxidative stress [61–63]. More recently, Thao et al. [51] suggested a possible link between NO and ET through MAPKs in plants exposed to HM stress. Accumulation of ET reduced NO levels and promoted the Cd-induced senescence processes in *Pisum sativum* [64]. Similarly, integration of ET, NO, PA, and MAPKs pathways improved tolerance in young *Glycine max* seedlings against short-term Cd stress [52]. The potential involvement of ETHYLENE INSENSITIVE2 (EIN2) in improving resistance against lead (Pb) stress has also been demonstrated in *Arabidopsis* [65]. Treatment of *Triticum aestivum* seedlings with Cd and PAs (spermine and putrescine) was found to induce NO generation in

**3. NO-phytohormone cross talk under heavy metals stress**

122 Phytohormones - Signaling Mechanisms and Crosstalk in Plant Development and Stress Responses

its availability in plants [45].

of metal and duration of stress [52].

roots leading to root growth inhibition [66].

Interplay between NO and GA has been reported to influence a wide spectrum of physiological processes, including seed germination, primary root growth, and inhibition of hypocotyl elongation [8, 29]. Interaction of NO with GA was observed to promote apical root growth in *Triticum aestivum* roots exposed to aluminum (Al) toxicity [50]. Contrary reports of Zhu et al. [74] showed that GA mediated alleviation of Cd toxicity in *Arabidopsis* was linked to reduction of Cd-induced NO accumulation and suppression of up-regulation of *IRT1*. Antagonistic relationship between NO and GA was also reported by Wu et al. [75] who noticed that NO accumulation inhibited the stimulatory effect of GA on primary root growth under low phosphorous (P) conditions. They suggested that GA interacts with NO and P pathways on DELLA-SLY module. Studies have shown the positive interaction between NO and CK, for example, Shen et al. [76] found that NO deficiency inhibited the activation CK-induced gene CYCLIN-D3;1 (CYCD3;1), which in turn promoted callus initiation from somatic plant tissues. Recent reports of Liu et al. [45] revealed that CK directly interact with NO to reduce endogenous NO levels in *Arabidopsis* implying the protective role of CK against nitrosative stress; however, no study has yet provided definitive evidence for NO and CK interaction under metal stress.

Combined NO and SA application was observed to counteract the toxic effects of Ni in *Brassica napus* through enhanced chlorophyll contents and reduced lipid peroxidation and proline accumulation [77]. Similarly, NO and SA increased Cd contents in cell walls of Cd-stressed *Arachis hypogaea* that reduced the distribution of Cd to organelles [78]. In recent years, accumulating evidence indicates the involvement of BRs induced NO production in root architecture and development [79]. Choudhary et al. [80] found that BR induced increased NO production promoted ABA synthesis that alleviated the toxic effects of Cu in *Raphanus sativus* seedlings.

Cross talk between plant hormones and NO is also considered critical for Fe-deficiency signaling [81]. Evidence obtained in *Arabidopsis* suggests interplay between ET and NO for upregulation of genes (A*tFIT*, *AtbHLH39*, *AtFRO2*, *AtNAS1*, *AtNAS2*, *AtFRD3*, *AtMYB72*) related to Fe-deficiency [82]. Although, it is well reported that enhanced NO generation helps to maintain root growth under Cd stress [83]; however, the exact role of NO under excess Fe conditions is rudimentary and demands further investigation.
