5. Interaction of SA with other signaling molecules

plants treated with 1 mM SA exhibited higher growth and improved physiology compared to

Salicylic acid is the most studied phytohormone regarding its role in oxidative stress. Due to its multifarious actions, it has been found very effective in detoxifying ROS in plant cells (Figure 2). According to Janda and Ruelland [144], SA-induced tolerance to abiotic stresses such as chilling, heat, heavy metals, osmotic stress, and salinity is involved in activation of the stress-induced antioxidant system. It has been demonstrated that SA could significantly improve both photosynthesis parameters and antioxidant defense system in conferring salt stress tolerance in V. radiata [17]. In O. sativa, exogenous SA significantly reduced the oxidative burst by reducing

upregulation of antioxidant defense enzymes (SOD, POD, CAT, APX, GR, and GST) and efficient GSH pool. The positive role of SA is mostly dependent on their dose and application methods [145]. Other study also proved that SA-mediated antioxidative defense system was dependent on the concentration used and the method of application [146]. In T. aestivum, 0.25 mM SA resulted in marked increase in the antioxidant enzyme activities (SOD, CAT, POD, GPX, APX, and GR), while the treatment with 2.5 mM SA resulted in a decrease in the activities under water stress [146]. A lower dose of SA also maintains higher AsA pool which in turns significantly scavenged the ROS. This ROS detoxification induced by SA also associated with improved photosystem II (PSII) efficiency. Belkadhi et al. [147] showed that Linum usitatissimum plants showed a lower amount of lipid and protein oxidation and membrane oxidation under Cd stress when pretreated with SA. This protection was availed by the enhanced activities of SOD, GPX,

reducing antioxidant power (FRAP). Increases in MDHAR, DHAR, GR, GPX, and CAT activities 53, 64, 49, 82, and 65% were noticed in PEG-treated B. juncea seedlings when sprayed with 50 μM SA [44]. SA-induced upregulation of antioxidant enzymes caused 32 and 26% decrease in MDA and H2O2 content compared to drought (20% PEG, 48 h) alone. One of our research results showed that exogenous SA enhanced the activities of antioxidant enzymes and nonenzymatic antioxidants under salt stress (200 mM NaCl, 48 h). Compared to salt stress alone, NaCl + SA resulted in 41, 107, 25, 37, 44, and 59% increases in MDHAR, DHAR, GR, GST, GPX, and CAT activities [40]. Salicylic acid supplementation also increased AsA and GSH content by 48 and 39%, respectively and enhanced GSH/GSSG ratio by 47% compared to salt stress alone. As a result, MDA and H2O2 contents decreased by 39 and 31% [40]. In Nitraria tangutorum SA mitigated salt-induced oxidative stress (evidenced by a marked reduction in MDA and H2O2 content) by upregulating the activities of SOD, POD, and CAT. The content of MDA in the 1.5 mM SA treated seedlings under 100–400 mM NaCl treatments declined to 2.27–3.59 fold of the control which was a clear sign of the reduction of oxidative stress [148]. However, some of the enzymes like APX activity was inhibited at higher concentrations (1.0 and 1.5 mM) of SAMDA content was measured with 1.5 mM SA applied, and the contents of MDA in the leaves of SAtreated seedlings under 100–400 mM NaCl treatments declined to only 2.27–3.59 fold of the

•� contents under herbicide exposure which was mainly due to the SA-mediated


nontreated one and subsequently showed better appearance under UV radiation [58].

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

4. Salicylic acid and ROS detoxification

H2O2 and O2

and APX as well as the 2,2<sup>0</sup>

control.

Salicylic acid not only exerts its positive effect independently but also interacts with other signaling molecules, phytohormones, and other phytoprotectants. These interactions show different signaling events and ultimate protection to plants from stress-induced damages (Figure 3). In grapevines, Wang and Li [102] showed improved Ca2+ homeostasis and associated antioxidant defenses under heat and cold stress-regulated by SA. When plants were treated with exogenous SA, they showed enhanced PM-Ca2+ATPases and V-Ca2+ ATPases activities. Moreover, Ca2+ precipitates were shown on the inner side of the plasma membrane, and less were in intercellular spaces and the vacuole. However, in SA treated plants Ca2+ precipitates were in vacuoles, and few were on the inner side of the plasma membrane. Ca2+ precipitates in chloroplasts were bigger even after heat or cold stress. Importantly, SA treatment caused enhancement of the activities of APX, GR, and MDHAR, which efficiently reduced the lipid peroxidation and relative EL (REL), which concluded that exogenous SA could mitigate oxidative stress by maintaining Ca2+ homeostasis under extreme temperature stress [102]. In O. sativa, Wang et al. [145] reported that SA treatment downregulated ABA genes more in cultivar XS 134, which correlated with the enhanced tolerance to quincloracinduced oxidative stress. Application of SA had obvious effects on all of the ABA-related genes and inhibited the expression of OsABA8ox1, OsABA8ox2, OsABA8ox3, OsNCED1, OsNCED2, and OsNCED3 as compared to quinclorac stress alone. Since overproduction of ABA and ROS is highly associated this downregulation protected the plants from herbicide-induced damages. Wang et al. [145] also reported SA-induced inhibition of ABA synthesizing enzymes. Leslie and Romani [149] reported that SA inhibited ethylene formation which triggered biosynthesis of ABA under stress conditions [150]. In the adventitious roots of Panax ginseng, SAinduced enhancement of the activities of NADPH oxidase, SOD, CAT, POD, and APX was evident while no significant effect on AsA and GSH content were observed [151]. These effects were mostly NO-dependent and it was also observed that SA-induced the generation of NO. They revealed that at lower concentration (100 μM) SA was highly effective in inducing the

Figure 3. Interaction of SA with other signaling molecules to elicit defense responses in plants.

accumulation of NO, O2 • and it took part in stress signaling. Interactive effects of SA and NO were studied in mitigating osmotic stress (0.4 MPa) in T. aestivum. It was observed that osmotic stress induced chl degradation and membrane instability, and H2O2 generation and lipid peroxidation were effectively reduced by exogenous application of SA or SNP, which was associated with the enhancement of antioxidant defense. However, pretreatment of plants with methylene blue (MB; as a guanylate cyclase inhibitor) reversed or reduced the protective effects of SA and SNP suggesting that the protective effects were likely attributed to NO signaling. They also concluded that NO may act as downstream of SA signaling in the reduction of induced oxidative damage [152]. SA-mediated H2O2 signaling and subsequent Cd stress tolerance was revealed in L. usitatissimum [147]. Seedlings pretreated with 250 or 1000 μM SA resulted in enhanced production of H2O2 because of inhibited CAT activity. Although the control plants with SA pretreatment showed significant (1.2 fold) increase in H2O2, this level is remarkably lower when compared with Cd alone and Cd+SA. These results indicated that SA could regulate the Cd-induced oxidative stress because Cd-treated seedlings primed with SA exhibited a higher level of total antioxidant capacities and increased activities of H2O2-detoxifying enzymes [147]. Exogenous SA application was found to activate GSH synthesis in B. juncea and B. napus and showed enhanced protection against drought- and saltinduced oxidative damages [44, 40].

#### 6. Conclusions and perspectives

Salicylic acid plays an important role in the regulation of growth and physiology in relation to the abiotic stress responses of plants. The SA was found to be effective in the different form of application foliar spray/incorporation with growing media depending upon plant species. The low concentration of SA showed advantageous effects in abiotic stress tolerance of plants. In contrast, the high concentration of SA showed toxic effects. Thus, both the concentration and application method of SA are critical to obtaining its best effect on different plant species. In the biosynthesis pathway of SA, there are unknown steps and enzymes which should be discovered. The catabolism and the further fate of transformed product of SA are not known clearly. How SA interacts and being regulated by the cross-talk in harmony with other phytohormones and plant growth regulators working (auxins, cytokinins, gibberellins, ethylene, jasmonates, brassinosteroids, etc.) and other signaling molecules (NO, H2O2) were not studied extensively. SA-mediated defense networks and insights into the cross-talk of SA with other defense-signaling pathways should be revealed. An integrated approach combining the knowledge of genetics, molecular biology, biochemistry, genomics, and bioinformatics techniques is a useful tool to study the functioning of SA in plants. Clear understanding of the biosynthesis and catabolic pathway and other unanswered question are vital to exploit SA as a potent phytoprotectant molecule to improve abiotic stress tolerances.

#### Acknowledgements

We are highly thankful to Mazhar Ul Alam, Laboratory of Plant Stress Responses, Faculty of Agriculture, Kagawa University, Japan, for his critical reading and formatting of the manuscript draft. The first author acknowledges Japan Society for the Promotion of Science (JSPS) for funding in his research. We are also highly thankful to Mr. Md. Mosfeq-Ul-Hasan, Zhejiang University, Hangzhou, China, for providing us several supporting articles. As page limitation precluded us from citing a large number of studies, we apologize to those whose original publications are therefore not directly referenced in this chapter.

### Author contributions

accumulation of NO, O2

induced oxidative damages [44, 40].

Acknowledgements

6. Conclusions and perspectives

• and it took part in stress signaling. Interactive effects of SA and NO

were studied in mitigating osmotic stress (0.4 MPa) in T. aestivum. It was observed that osmotic stress induced chl degradation and membrane instability, and H2O2 generation and lipid peroxidation were effectively reduced by exogenous application of SA or SNP, which was associated with the enhancement of antioxidant defense. However, pretreatment of plants with methylene blue (MB; as a guanylate cyclase inhibitor) reversed or reduced the protective effects of SA and SNP suggesting that the protective effects were likely attributed to NO signaling. They also concluded that NO may act as downstream of SA signaling in the reduction of induced oxidative damage [152]. SA-mediated H2O2 signaling and subsequent Cd stress tolerance was revealed in L. usitatissimum [147]. Seedlings pretreated with 250 or 1000 μM SA resulted in enhanced production of H2O2 because of inhibited CAT activity. Although the control plants with SA pretreatment showed significant (1.2 fold) increase in H2O2, this level is remarkably lower when compared with Cd alone and Cd+SA. These results indicated that SA could regulate the Cd-induced oxidative stress because Cd-treated seedlings primed with SA exhibited a higher level of total antioxidant capacities and increased activities of H2O2-detoxifying enzymes [147]. Exogenous SA application was found to activate GSH synthesis in B. juncea and B. napus and showed enhanced protection against drought- and salt-

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

Salicylic acid plays an important role in the regulation of growth and physiology in relation to the abiotic stress responses of plants. The SA was found to be effective in the different form of application foliar spray/incorporation with growing media depending upon plant species. The low concentration of SA showed advantageous effects in abiotic stress tolerance of plants. In contrast, the high concentration of SA showed toxic effects. Thus, both the concentration and application method of SA are critical to obtaining its best effect on different plant species. In the biosynthesis pathway of SA, there are unknown steps and enzymes which should be discovered. The catabolism and the further fate of transformed product of SA are not known clearly. How SA interacts and being regulated by the cross-talk in harmony with other phytohormones and plant growth regulators working (auxins, cytokinins, gibberellins, ethylene, jasmonates, brassinosteroids, etc.) and other signaling molecules (NO, H2O2) were not studied extensively. SA-mediated defense networks and insights into the cross-talk of SA with other defense-signaling pathways should be revealed. An integrated approach combining the knowledge of genetics, molecular biology, biochemistry, genomics, and bioinformatics techniques is a useful tool to study the functioning of SA in plants. Clear understanding of the biosynthesis and catabolic pathway and other unanswered question are vital to exploit SA as a

We are highly thankful to Mazhar Ul Alam, Laboratory of Plant Stress Responses, Faculty of Agriculture, Kagawa University, Japan, for his critical reading and formatting of the manuscript

potent phytoprotectant molecule to improve abiotic stress tolerances.

M.H. performed literature reviews and drafted the manuscript; K.N., T.I.A. and T.F.B., M.I. and M.H. contributed the review for literature research; M.F. and H.O. reviewed the manuscript and approved the final draft.

#### Conflicts of interest

The authors declare no conflict of interest.

#### Abbreviations


