**4. ROS generation and removal in the plants**

Reactive oxygen species (ROS) are a broad term that includes the radical and nonradical form of species, formed due to incomplete oxygen metabolism. Radical species include superoxide radical (O2•\_), hydroxyl radical (•OH), alkoxyl (RO•) and peroxyl (ROO•) while non- radical species contains hydrogen peroxide (H2O2), singlet

#### *ROS Regulation Mechanism for Mitigation of Abiotic Stress in Plants DOI: http://dx.doi.org/10.5772/intechopen.99845*

oxygen (1O2), ozone (O3), and hypochlorous acid (HClO). Oxygen is a fundamental element found in the Earth's crust that evolved billions of years ago. Oxygen molecules (O2) are not only crucial for metabolism and respiration but also support life forms on the Earth. O2 are mainly evolved through the photosynthetic activities of cyanobacteria in ancient times. ROS are partially reduced or activated derivatives of oxygen molecules that are highly reactive and toxic and can cause potential damage to the plants which includes cellular destruction, damage to plant metabolism and growth along with damage to DNA, RNA, proteins, and lipids. Plants perform various metabolic processes viz., namely photosynthesis, respiration which leads to the production of reactive oxygen species in various cell organelles like mitochondria [7], peroxisomes [8], chloroplasts [6], etc. They are an unavoidable phenomenon that leads to the production of oxidative stress in plants. ROS can also be produced during abiotic and biotic stress responses in plants. Besides this, the presence of free metals (Fe, Cu, Mn) derived from the metallo-protein complex phenomenon also contributes to ROS production. The production of ROS is stimulated by many factors namely physiological responses in the plant cell organelles, hormonal signaling, pathogen attack, gravitropism which produces free radicals inside plants [101]. The stoichiometry of ROS reveals that oxygen contains two unpaired electrons in their outermost shell having similar spin quantum numbers. However, oxygen molecules can accept a single electron at a time in its outermost orbit due to spin restriction, resulting in the formation of ROS which is highly reactive and active in subsequent reactions [102]. ROS are atoms or groups of atoms that possess at least one unpaired electron. Oxygen is an indispensable part of aerobic reactions in the plant system and molecular reduction of O2 leads to the formation of reactive oxygen species various cell organelles which are highly reactive as of molecular oxygen. Photosynthesis is a crucial metabolic process performed by the plants in the chloroplast of plant cells due to the localization of photosynthetic apparatus in the chloroplast. Although the photosynthetic process is highly influenced by the generation of ROS (O2.-&1O2), the formation of superoxide radicals is associated with PSI. The photolysis of water molecules is a crucial phenomenon in the PSII system of photosynthesis which produces O2 thus favoring superoxide radical formation reaction in PSI of photosynthesis. Also, auto-oxidation of Ironsulfur protein results in O2.- production in the subsequent process due to abundant Fred and low NADP. Furthermore, reduced Fd reacts with superoxide radicals to form H2O2 in the illuminated chloroplast [103]. However, the regulation of ROS production during photosynthetic processes has been enunciated in several studies [53, 58, 104]. Singlet oxygen is also produced in PSII during photosynthetic processes. However, the root and stems of rice plants mainly produced O2-Which might relate to their subsequent environment for adaptation [105]. Additionally, superoxide radicals can be generated during PSII by auto-oxidation of PSII electron acceptors and PQ [106]. The ROS formation in illuminated chloroplast occurs mainly due to stress conditions followed by the closing of stomata. The partial reduction of O2 molecule in the respiratory chain occurs in chloroplast which consists of NADPH dehydrogenase and terminal oxidase is termed as chlororespiration. This phenomenon is also a major source of ROS production in the chloroplast. The peroxisomes also mediate the formation of O2.-through ETC using NADH as an electron donor. Peroxisomes are single membrane-bound organelle that performs certain major functions in the plants like fatty acid β- oxidation, regulation of glyoxylate cycle, photorespiration, metabolism of ROS & ureides, etc. [107–109]. Additionally, peroxisomes also regulate the generation of ROS via various metabolic functions. For instance, H2O2 production in the peroxisomes facilitated the regulation of the photosynthetic carbon oxidation cycle in C3

plants. During the carbon oxidation cycle, oxygenation of RuBP (mediated by RuBisCO) regenerates NADP+ and harbors a major sink of electrons which in turn prevents photoinactivation of PSII in the case when CO2 concentration is lacking. Due to which RuBisCO stimulates oxygenation in place of carboxylation as temperature elevates. The glycolate thus generated by oxygenation of RuBisCO suffers oxidation upon translocation to peroxisomes from chloroplast produces H2O2 as a by-product in the cells [102, 110]. Photorespiration is a metabolic process that occurs in chloroplast, mitochondria & peroxisomes. It includes phosphoglycolate metabolism which involves light-dependent O2 uptake & CO2 release with peroxisomal glycolate oxidase generating (H2O2) in the cells. The mitochondrial metabolism generated a considerable amount of ROS like H2O2, hydroxyl radicals, superoxide, etc. In the plants, mitochondria regulate aerobic respiration includes ETC (electron transport chain), which formulates ROS production in the mitochondrial membrane. However, the mitochondria bestow the limited ROS production in plants possibly due to the presence of alternative oxidase (AOX) that catalyzes the tetravalent reduction of O2 by ubiquinone [10]. The series of metabolic reactions in mitochondria leads to the formation of ROS inside the organelle. The flavoprotein region of NADH dehydrogenase encourages the production of O2- anions during mitochondrial electron transport (MET). One of the effective inhibitors of MET antimycin A enhances the ROS production by blocking electron flow after ubiquinone and the reduced ubiquinone undergoes auto-oxidation by contributing electron to O2, forming O2- [111]. Additionally, researchers studied that ubiquinone also contributes to H2O2 production in MET [112, 113]. Several mitochondrial enzymes like aconitase and 1-galactono-γ lactone dehydrogenase (GAL) also contribute to ROS generation. Furthermore, the O2 also gets converted into stable form H2O2 by the mitochondrial form of SOD (Mn-SOD). H2O2 is further transformed to (OH.) through Fenton reaction which is removed by ascorbate- glutathione cycle enzyme in plant system. Such OH molecules are liable to mutations in ETC of the mitochondrial genome. ROS generation in mitochondria also possesses negative effects on proteins by oxidation, cleavage, degradation of backbones [106]. Mitochondrial dysfunction due to excessive ROS production under unfavorable circumstances induces PCD (programmed cell death) and necrosis in the plants. The respiratory burst oxidase homologue (RBOH) synonymous with membrane-bound NADPH oxidase (NOX) in the plants also contributes to O2. formation through electron through from intracellular NADPH across the plasma membrane to O2 in apoplast [103]. NADPH oxidase has a well-established role in stress responses in plants. Neill et al, 2002 studied that RBOH- dependent O2. generation enunciates lipid peroxidation, PCD. NADPH oxidase induces membrane damage, favors oxidative burst, and reduces plant metabolic and growth-related activities under certain undesirable conditions. The plant cell wall is a site of redox reactions which enables the H2O2- dependent reactions and consists of malate dehydrogenase and NADH oxidase. NADPH-dependent microsomal electron transport is also a potential source of superoxides in the plant system. O2– formation in microsomes is mainly progressed by auto-oxidation of cytochrome P-450 reductase and/or auto-oxidation of oxycytochrome –P-450 complex [114]. Thus, the plant system involves efficient methods of ROS production in different responses. Different cellular compartments enunciate constant ROS production as a byproduct of redox and aerobic reactions. It is interesting to know that under favourable conditions plant maintains redox homeostasis and counteract with ROS production strategies within its system. The system of higher plants is complex and posses various regulatory processes. It is important to understand that controlled ROS generation and oxidative

#### *ROS Regulation Mechanism for Mitigation of Abiotic Stress in Plants DOI: http://dx.doi.org/10.5772/intechopen.99845*

burst can be a fundamental part of the plant signaling and defense mechanism. Future acknowledgments and researches should be directed towards the detailed study of ROS production in various aspects in plants with its signaling implications with references to various molecules present in the plant system. Plants possess a peculiar ROS scavenging system to maintain ROS homeostasis and redox signaling in their system during oxidative stress. However, disrupted protective mechanisms in response to oxidative burst may affect ROS-mediated redox homeostasis and cause cell death in the plants [11]. It is well-acquainted fact that plant produces a considerable amount of ROS during various metabolic processes in several cell organelles like chloroplast, mitochondria, plasma membrane and many more. They are an inexorable part of plant metabolism which play important role in redox signaling under environmental stresses [115]. To accomplish ROS removal from plant system plants consists of several ROS scavenging system which can be categorized into enzymatic and non-n enzymatic defense mechanism. Plants contain various antioxidant enzymes to mediate ROS scavenging mechanism which includes superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), catalase (CAT), monodehydroascorbate reductase (MDHAR or MDAR), dehydroascorbate reductase (DHAR or DAR) and glutathione reductase (GR) (**Figure 2**). These antioxidant enzymes ensure plant survival by minimizing the deleterious effect of ROS and prevent its overaccumulation. SOD (1.15.1.1) is a ubiquitous enzyme that plays a

#### **Figure 2.**

*Enzymatic and Non enzymatic Antioxidant mechanism to defend oxidative stressEnzymatic and non-enzymatic antioxidants in algae.ASC(Ascorbate),APX,(Ascorbate peroxidase),CAT Catalase, DHA Dehydroascorbate, GSH (Glutathione), GR Glutathione reductase , GSSG (Glutathione disulfide ),MDHA (Monodehydroascorbate ),SOD (Superoxide dismutase ),DHA (Dehydroascorbate).*

significant role in plant protection against oxidative stress. It catalyzes the dismutation of O2—to O2 and H2O. SOD has several isoforms and can be categorized as FeSOD, MnSOD, NiSOD, Cu/Zn SOD based on metal cofactors associated with the enzymes. The Arabidopsis genome contains three FeSOD (FSD1, FSD2, FSD3), one MnSOD (MSD1), and three Cu/Zn SOD (CSD1, CSD2, CSD3) type of genes [116, 117]. Similarly, the tomato genome consists of four Cu/Zn SOD, three FeSOD, and one MnSOD [118]. SOD gene family also have been discovered in many plant species like Musa acuminate, Sorghum bicolor, Populus trichocarpa, potato, pea, wheat, etc. However, transgenic approaches have also been described to study SOD responses in plants [10]. SOD isozymes have also been compartmentalized into mitochondria [119], peroxisomes [9], cytosol [116, 120], thylakoids [116, 121]. In plants, SOD is found in roots, leaves, fruits, and seeds where it functions significantly in the environment and oxidative stresses [122], photooxidative stress [123, 124], lateral root growth [119], germination [120], chloroplast development and flowering [121, 125]. Catalases (CAT) (E.C. 1.11.1.6) are a versatile antioxidant that helps in ROS scavenging mechanism in plants. They are ion containing homotetrameric proteins that catalyze the decomposition of H2O2 to H2O and O2 during the photorespiration process along with detoxification of H2O2[126]. Catalases are involved in antioxidant defense mechanism have been enunciated in many studies [127–132]. They also mediate in various physiological processes [45, 133–135]. The oxidative stress in plant cells can be maintained by enzymes of ascorbate- glutathione cycle. Ascorbate peroxidase (APX) (E.C.1.11.1.11) is another class of antioxidant enzyme that plays a vital role in scavenging H2O2 in chloroplast and cytosol in the plants. They are categorized into various forms based on their localization which is mainly chloroplast stromal soluble form (sAPX), chloroplast thylakoid bound form (tAPX), cytosolic form (cAPX) and glyoxisome membrane form (gmAPX) [136, 137]. They are heme-containing peroxidases possessing nine putative APX genes identified in Arabidopsis in cytosolic, chloroplast and peroxisomal regions of plant cells [138, 139] and sAPX in mitochondria [140]. They detoxify H2O2 through electron transfer from ascorbate to form monodehydroascorbate (MDHA). APX possesses several metabolic functions in H2O2 scavenging, plant responses to environmental stress, photoprotection, and plant development [115]. Another enzyme of ascorbate- glutathione cycle MDHAR (E. C.1.6.5.4) catalyzes the reverse reduction of MDHA to ascorbate in the presence of NAD(P)H [141] Foyer & Noctor, 2011). MDHAR is mainly localized in the cytosol, peroxisomes, mitochondria, and chloroplast. MDHAR is involved in stress tolerance, plant physiological processes, senescence, interaction, with endophytes has been explored in various studies [115, 142, 143]. DHAR is another enzyme that brings about the regeneration of ascorbate from DHA. DHARs (E.C.1.8.5.1) are monomeric enzymes that are identified in the Arabidopsis genome as DHAR1 and DHAR2 (cytosol), DHAR3 (chloroplast) [144]. Similar to APX, DHAR is also involved in the regulation of defense against environmental stress in various species [145, 146]. Glutathione reductase (GR) (E.C.1.6.4.2) is another potent enzyme mainly localized in chloroplast, mitochondria and cytosol. It catalyzes the reduction of glutathione and contains FAD-binding domain & NADPH- binding domain which carries out an enzymatic activity. Isozymes of GR have been widely studied in Arabidopsis which plays a vital role in various plant physiological responses [115].

The non-enzymatic antioxidant defense mechanism includes several low molecular mass ROS scavenging molecules like glutathione, ascorbic acid (AsA), flavonoids, carotenoids, tocopherols, alkaloids which aid in the removal of H2O2, singlet oxygen, and other ROS molecules. The antioxidant defense mechanism of AsA during

#### *ROS Regulation Mechanism for Mitigation of Abiotic Stress in Plants DOI: http://dx.doi.org/10.5772/intechopen.99845*

oxidative burst has been well acquainted through several studies. AsA (commonly known as Vit C) is water-soluble, localized in many plant cell organelles stimulates the quenching superoxide hydroxyl radicals and singlet oxygen produced during oxidative stress. Despite these, it also reduces H2O2 to H2O via ascorbate peroxidase reaction [147]. AsA regulates antioxidant defense mechanisms in response to various environmental stresses [148]. The non-enzymatic antioxidant system functions along with an enzymatic system to counteract the negative effect of ROS in plants. Reduced glutathione (GSH) is another class of low M.W thiol tripeptide antioxidant molecule commonly dominated in the cytosol, ER, mitochondria, chloroplast, vacuoles, peroxisomes & apoplast. GSH mediates multiple functions in plants. It plays a vital role in plant physiological functions like cell differentiation, growth, senescence, and many more. Precisely, it is also known for its antioxidant defense system in oxidative stress. It scavenges H2O2, 1O2, OH., O2.–, and reduces them to produce GSSG as a byproduct. GSSG can also be generated through GSH. GSH plays a crucial part in regenerating AsA via the ascorbate- glutathione cycle. GSSG gets converted to GSH through denovo synthesis or by GR. Similar to glutathione another potential antioxidant molecule also includes proline, amino acids, alkaloids, polyamines, terpenes, amines, phenolics like compounds that scavenge ROS in plants. Carotenoids, on the other hand, are a group of lipophilic antioxidants that are present in a wide variety of organisms including plants. They have a well-established role in photosynthesis and protect photosynthetic machinery in response to ROS production. They scavenge 1O2, thus preventing generation by reacting with 3Chl\* and excited Chl (Chl\*), regulates the xanthophylls cycle. Similar to carotenoids, α- tocopherol is also a protector of the cell membrane in response to ROS production. α- tocopherol quench excess energy, safeguard lipids, and scavenges ROS formed during photosynthesis. It usually reacts with lipid radicals RO., ROO. And RO\* at membrane- water interface and gets reduced to TOH. Which is then interacts with GSH & AA [149]. Flavonoids like flavonols, flavones, isoflavones, and anthocyanins are diverse in the plant kingdom and also plays a crucial role in the various physiological process mainly pigmentation in flowers, fruits, and seeds. They mitigate the negative effects of ROS produced in plants during photosynthesis. Additionally, they also scavenges1O2 along with repairing chloroplast membrane [150].
