**3. Oxidation Window**

systemic cross-talk between the ROS and phytohormones during the transition period

**Keywords:** dormancy, germination, metabolism, oxygen radicals, signaling, mecha‐

After pollination (double fertilization), the typical diploid embryos are covered by the triploid endosperm and the diploid testa. The triploid endosperm consists of nutritive tissues and living cells, while the diploid testa includes seed coat, maternal tissue, and dead cells. Seeds are the vital component allowing embryo dispersion and its consequent development into mature plants [1]. The seeds of monocots and dicots differ in their structure and method of emergence [1, 2]. However, here we comprehensively focus on the signaling pattern of the reactive oxygen species (ROS) and its interaction during the germination and dormancy

Although the dispersal of seeds is absolutely dependent on various cues such as vitality, genotype, hardness, moisture, air, temperature, light, and duration of seed storage, endosperm weakening is one of the key factors that determine the protrusion of radicle. The term "coatassociated dormancy" refers to the mechanical constraint that can impair germination, while

After imbibition, the weakening of endosperm is dependent on the gas exchange/respiration. Loosening of the endosperm was suggested to be influenced by the proper localization of ROS and its fine regulation by the antioxidant systems [4, 5]. The proper reduction/oxidation of the ROS (redox homeostasis) plays a key role in the transition from quiescence to active state [6]. Gibberellins (GAs) are involved in the promotion of the endosperm weakening and, on the other hand, abscisic acid (ABA) at least partly inhibits this process either directly or indirectly [2, 3, 7]. Induction/inhibition of the genes responsible for the endosperm weakening is controlled by the ROS-GA-ABA [7–11]. Substantially, cross-talk of other phytohormones, such as ethylene, brassinosteroids and auxins, with GA and ABA were reported to be inevitable for

Although research in seed biology has reached significant advancements, apparent continuum still lies in the mechanisms underlying germination and dormancy, which needs to be disclosed. In this chapter, the heuristic network on cross-talk between the ROS and phytohor‐

To fulfill the higher energy requirement during the transition period (from quiescent to active state) of the seed, cellular respiration is rapid, high, and synchronized with the mitochondrial

mones involved in the release and/or induction of dormancy has been discussed.

"embryo dormancy" is characterized by the embryo failure to develop [1–3].

of quiescent seeds into the metabolically active organisms.

76 New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology

nism

**1. Introduction**

condition.

the seed development [12].

**2. Seed Respiration**

During the embryogenesis and the seed-filling process, seeds possessed maximum water content [17]. Subsequently, dramatic water loss takes place in the postmaturation stage [2]. According to the recent reports, ROS do not play a detrimental role during development under controlled conditions [18]. The ROS-mediated signaling is majorly involved in the endosperm weakening, mobilization of seed reserves, programmed cell death (PCD), and also protection against the pathogens [4]. Hence, it can be ascertained that the ROS cannot be simply consid‐ ered as a hazardous material. Controlled production of ROS and ROS-related molecular interactions represent key factors in various central components of plant biology [16]. In the nondormant seeds, O2 − and H2O2 radicals were uniformly distributed within the radicle, while in dormant seeds irregular patterns were observed. Only seeds with a proper redox homeo‐ stasis display nondormant phenotype [7, 11]. Success of the seed germination is apparently associated with the equilibrium between the ROS and its scavenging antioxidant system [1, 4– 6]. Uncontrolled generation of ROS is extremely harmful and can lead to several lethal effects. Meanwhile, the tight control over the ROS helps in various developmental processes including germination. This process is generally termed as 'oxidation window' [19].

#### **3.1. Quiescent seed**

Quiescent seeds, characterized by low moisture content (5–15%), do not possess active metabolism. During the late embryogenic state, seeds are actively involved in the storage of reserves, while enzymatic activities are gradually decreased. However, during the storage, lipid peroxidation (LPO) occurring on the polyunsaturated fatty acids (PUFA) in the cell membrane constantly releases the ROS [2, 3]. Longevity of seeds depends on the free radicals generated by the LPO. In the dried condition, ROS are released from polyunsaturated fatty acids by LPO [4]. The free radicals focused on the H-atom in the methyl group of lipids. The single cleavage leads to the release of LOO•, and the double cleavage leads to the release of LOOH [16]. Depending on the aging extremity, ROS affects the viability of the seed. Most of the enzymatic activities are arrested in the dried state of the seed. Damages caused by LPO cannot be retained during the transition from a quiescent to an active state [19, 20]. From the epigenetic study of Nakabayashi et al., it can be suggested that more than 12,000 stored mRNA species or transcripts were detected in the desiccated seeds of *Arabidopsis*. This number is almost a half of the whole genes present in *Arabidopsis*. Moreover, promoters of the highly expressed genes overrepresented the abscisic acid-responsive elements (ABREs) containing motif ACGT that are sufficient for the ABA-induced transcription [4, 21]. During the increase in a desiccation rate, the accumulations of late embryogenesis abundant (LEA) proteins are also increased to enhance the tolerance against water loss. Among various LEA proteins identified, group-2 LEA-dehydrins are highly involved for the desiccation tolerance [22]. Jiang and Kermode reported that nondormant and dormant phenotypes of the seeds were defined by their desiccation tolerance level. Seed storage proteins play important roles during the dehydration processes. If the desiccation process is imposed prematurely or deterioration takes place and then synthesis of storage proteins will be terminated. Consequently, seeds become more sensitive to stress and lose their vigors [23]. The processes of maturation drying are associated with the ability of seed for germination.

#### **3.2. Imbibed seed**

In general, germination normally begins with the imbibition of seeds by 70–80% of water. El-Maarouf-Bouteau and Bailly reported that high levels of ROS are accumulated during the imbibition phase [5]. This might be due to the resumption of metabolically active sites such as mitochondria, chloroplasts, peroxisomes, glyoxysomes, and plasma membranes. The mito‐ chondrial electron transfer chain (ETC) was considered as a primary source for the ROS (O2 − ). Foyer et al. reported that 2–3% of oxygen from the mitochondria was the source of O2 − and H2O2. In addition, chloroplast, a vital site for photosynthesis, and ETCs from the photosystems, such as PSI and PSII, produce O2 *−* , 1 O2, and H2O2. Meanwhile, the mobilizations of the lipids stored in the embryo carried out by the glycolate oxidase are another source of O2 *−* and H2O2. Due to the catabolism of lipids and purines in the glyoxysomes and peroxisomes, the release of O2 *−* , H2O2, and nitric oxide (NO) is inevitable [24]. The H2O2 is majorly released in the peroxisomes during the conversion of glyoxylate catalyzed by the glyoxylate oxidase. Subsequently, fatty acid β-oxidation by the flavin oxidase generated the •OH and NO. Meanwhile in the peroxisome, xanthine conversion to uric acid, catalyzed by the xanthine oxidase, releases enormous amount of O2 *−* . Recent attention on the cell-wall-dependent peroxidases, oxalate oxidases and NADPH oxidases, and their involvement in the transfer of electrons indicate the plasma membrane to be another important site for the ROS synthesis [25]. The NADPH oxidase, amine oxidase, chytochrome p450, cell wall peroxidase, and germin-like oxalate oxidases disperse the H2O2 from cell to cell [4, 24].

The hydrated state of seeds allows the longer shelf life H2O2 to reach the targets distant from the production sites [16]. As mentioned earlier during the unfavorable condition, ROS lead to the breakdown of essential macromolecules such as lipids, nucleic acids, proteins, and other deleterious activities [19]. In the favorable condition, the ROS stimulates the mobilization of reserves and selectively interact with the targets by oxidation. This oxidation triggers a genespecific signaling pathways and also activates the transcription factors (TFs) either directly or indirectly [15, 19, 20]. The cleavage of cell wall-polymers of endosperm can be correlated with the over-expression of cell wall-peroxidases [26]. During the putative shift from the desiccation to the germination state, exogenous application of optimal H2O2 increased the regulation of 113 genes and decreased the regulation of 62 genes in *Arabidopsis* [27]. Initial imbibition conditions determine the fate of the subsequent metabolic pathways that are required to complete seed germination.

#### **3.3. Temporal and spatial regulation of ROS accumulation**

The metabolically active sites are the source of ROS. As the range and action of ROS are limited by diffusion, ROS production source determines its molecular mobility and viscosity [28]. The rate of metabolic activity and the source of ROS production govern the process of seed development. Leymarie et al. reported that after imbibition, the ROS are first localized in the cytoplasm followed by the nucleus and lastly in the cell wall [29]. In the cytoplasm, ROS modulates the redox homeostasis which triggers the protein oxidation and mRNA synthesis is the first sign of seed germination process [30–32]. Antioxidant systems are concordantly involved in maintaining the ROS level. The fine tuning of the ROS is achieved by the direct or indirect interaction with the transcription factors of the genes responsible for the redox status. Finally, the NADPH oxidase located in the cell wall helps in cell-to-cell propagation [33]. In the dormant phenotype, ROS production is high and also scattered. In the dormant phenotype, the ROS is properly diffused from cytoplasm to nucleus and cell wall [19]. The role of ROS (either beneficial or deleterious) is dependent on its distribution. Therefore, the temporal and spatial accumulations of the ROS are inevitable for proper germination [15, 29].

#### **3.4. Protein carbonylation**

− ).

− and

and H2O2.

*−*

membrane constantly releases the ROS [2, 3]. Longevity of seeds depends on the free radicals generated by the LPO. In the dried condition, ROS are released from polyunsaturated fatty acids by LPO [4]. The free radicals focused on the H-atom in the methyl group of lipids. The single cleavage leads to the release of LOO•, and the double cleavage leads to the release of LOOH [16]. Depending on the aging extremity, ROS affects the viability of the seed. Most of the enzymatic activities are arrested in the dried state of the seed. Damages caused by LPO cannot be retained during the transition from a quiescent to an active state [19, 20]. From the epigenetic study of Nakabayashi et al., it can be suggested that more than 12,000 stored mRNA species or transcripts were detected in the desiccated seeds of *Arabidopsis*. This number is almost a half of the whole genes present in *Arabidopsis*. Moreover, promoters of the highly expressed genes overrepresented the abscisic acid-responsive elements (ABREs) containing motif ACGT that are sufficient for the ABA-induced transcription [4, 21]. During the increase in a desiccation rate, the accumulations of late embryogenesis abundant (LEA) proteins are also increased to enhance the tolerance against water loss. Among various LEA proteins identified, group-2 LEA-dehydrins are highly involved for the desiccation tolerance [22]. Jiang and Kermode reported that nondormant and dormant phenotypes of the seeds were defined by their desiccation tolerance level. Seed storage proteins play important roles during the dehydration processes. If the desiccation process is imposed prematurely or deterioration takes place and then synthesis of storage proteins will be terminated. Consequently, seeds become more sensitive to stress and lose their vigors [23]. The processes of maturation drying

78 New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology

In general, germination normally begins with the imbibition of seeds by 70–80% of water. El-Maarouf-Bouteau and Bailly reported that high levels of ROS are accumulated during the imbibition phase [5]. This might be due to the resumption of metabolically active sites such as mitochondria, chloroplasts, peroxisomes, glyoxysomes, and plasma membranes. The mito‐ chondrial electron transfer chain (ETC) was considered as a primary source for the ROS (O2

Foyer et al. reported that 2–3% of oxygen from the mitochondria was the source of O2

stored in the embryo carried out by the glycolate oxidase are another source of O2

*−* , 1

germin-like oxalate oxidases disperse the H2O2 from cell to cell [4, 24].

H2O2. In addition, chloroplast, a vital site for photosynthesis, and ETCs from the photosystems,

Due to the catabolism of lipids and purines in the glyoxysomes and peroxisomes, the release

*−*

peroxidases, oxalate oxidases and NADPH oxidases, and their involvement in the transfer of electrons indicate the plasma membrane to be another important site for the ROS synthesis [25]. The NADPH oxidase, amine oxidase, chytochrome p450, cell wall peroxidase, and

, H2O2, and nitric oxide (NO) is inevitable [24]. The H2O2 is majorly released in the peroxisomes during the conversion of glyoxylate catalyzed by the glyoxylate oxidase. Subsequently, fatty acid β-oxidation by the flavin oxidase generated the •OH and NO. Meanwhile in the peroxisome, xanthine conversion to uric acid, catalyzed by the xanthine

O2, and H2O2. Meanwhile, the mobilizations of the lipids

. Recent attention on the cell-wall-dependent

are associated with the ability of seed for germination.

**3.2. Imbibed seed**

of O2 *−*

such as PSI and PSII, produce O2

oxidase, releases enormous amount of O2

Seed vigor is mainly affected by the protein oxidation process such as carbonylation and decarbonylation. Protein carbonylation is the oxidation of proteins caused by the ROS, especially on the side chains of lysine, arginine, proline, and threonine [34]. Decrease in the carbonylation of proteins is known as decarbonylation [35]. Activation on the oxidation phase of pentose phosphate pathway (oxPPP) modulates the carbonylation of proteins. Modulation of redox potential in the glycolysis and oxPPP were observed during the release of dormancy [36]. The interaction or signaling of the ROS determines or fine tunes various translation and posttranslation processes during the seed development. Job et al. reported that in the dry seed, proteins, such as 12S-cruciferin subunits, aldose reductase and the LEA, undergo carbonyla‐ tion. After imbibition, protein carbonylation specifically targets glycolytic enzymes, mito‐ chondrial ATP synthase, chloroplastic ribulose carboxylase large chain, aldose reductase, methionine synthase, translation factors, and molecular chaperones [37]. The NADPH-oxidase also known as respiration burst oxidase homolog (rboh) plays an important role in the transfer of electrons from cytosolic NADPH or NADH to apoplastic oxygen and posttranslational modifications of proteins. In Arabidopsis, *AtrbohD* mutant showed reduced superoxide production and protein carbonylation in dry seeds. However, after imbibition the protein, oxidation level of *AtrbohD* mutant was slightly higher than wild [38]. The posttranslational modifications, especially mRNA oxidations, are governed by the ABA [32].

#### **3.5. Antioxidant enzymes**

As mentioned earlier, improper desiccation as well as storage increases the LPO and affects seed vigor. Increased production of ROS from the metabolically active sites during the transition from a quiescent to imbibition state could possibly cause stress. The deleterious effects of the ROS can be overcome by the proper antioxidant system. Both enzymatic and nonenzymatic antioxidants play a vital role in the maintenance of level of the ROS. Rather than complete alleviation, proper activation of antioxidant enzymes directs the ROS to the signaling process [24–27]. Muller et al. reported that ROS are important components in the endosperm weakening [26]. Exogenous application of H2O2 or menadione (to generate superoxide) to 3 day old maize seedlings enhances tolerance against the chilling stress [39]. Meanwhile, Pulido et al. found that nuclear localization of peroxiredoxin and thioredoxin prevents nucleic acids from oxidative damage occurring during the maturation and germination in wheat seeds [40]. The detoxification of H2O2 in the seed filling is catalyzed by the isoforms of catalase (CAT). The isoform CAT3 is involved highly in the early postpollination, whereas CAT1 and CAT2 isoforms play a crucial role during the seed development [41, 42]. Recently, Leymarie et al. clearly demonstrated the necessity of the ROS and the antioxidant enzymes for successful germination using mutant seeds. In the *Arabidopsis cat2-1* mutant, intracellular H2O2 and redox perturbation were increased. In case of the *vte1-1* mutant lacking a gene that encodes the tocopherol cyclase, an increase in the redox active biosynthetic intermediate was observed [29]. Tocopherol is generally involved in the protection of lipids from oxidation. Tocopherols, also called vitamin E, functions as terminators of a PUFA recyclable chain reaction. The tocopher‐ oxyl radical can be recycled back to the tocopherol by the reaction of ascorbate or other antioxidants [43]. The lack of the *vte1-1* function in the seeds releases lipid peroxy radicals. Although the *cat2-1* and *vte1-1* affect the seed germination to a certain level, plants lacking the *rbohD* gene can successfully complete germination (with time delay). The *rbohD* gene is involved in the conversion of the O2 and NADPH to form superoxide and plays a vital role in the cell-to-cell propagation of the ROS and generation of the •OH. The •OH is essential for the cell wall loosening of endosperm [29].

#### **3.6. Nonenzymatic antioxidants**

Nonenzymatic antioxidants that actively participate in the ROS equilibrium are ascorbate, glutathione, and preoxiredoxins [44–47]. Low moisture content decreases the molecular mobility and the accessibility of substrates for the catalysis of antioxidant enzymes [44]. Ascorbate plays a major role in the progression of cell cycle, cell growth, hormonal signaling pathways, and embryogenesis. Ascorbate content of the seed decreased the H2O2 by increasing the peroxiredoxins [45]. Nonenzymatic antioxidants also determine the protection of cells against the ROS, particularly at the desiccation stage. Tocopherol is involved in the prevention of membrane damages by the LPO during a prolonged seed storage [4]. Peroxiredoxins protect the nuclear integrity and prevent against the oxidative damages of DNA under high levels of •OH radicals [46]. Involvement of the ascorbate-glutathione cycle alone in the seeds could be another vast area, which needs to be discussed separately.

### **3.7. Interplay between ROS, GA, and ABA**

also known as respiration burst oxidase homolog (rboh) plays an important role in the transfer of electrons from cytosolic NADPH or NADH to apoplastic oxygen and posttranslational modifications of proteins. In Arabidopsis, *AtrbohD* mutant showed reduced superoxide production and protein carbonylation in dry seeds. However, after imbibition the protein, oxidation level of *AtrbohD* mutant was slightly higher than wild [38]. The posttranslational

As mentioned earlier, improper desiccation as well as storage increases the LPO and affects seed vigor. Increased production of ROS from the metabolically active sites during the transition from a quiescent to imbibition state could possibly cause stress. The deleterious effects of the ROS can be overcome by the proper antioxidant system. Both enzymatic and nonenzymatic antioxidants play a vital role in the maintenance of level of the ROS. Rather than complete alleviation, proper activation of antioxidant enzymes directs the ROS to the signaling process [24–27]. Muller et al. reported that ROS are important components in the endosperm weakening [26]. Exogenous application of H2O2 or menadione (to generate superoxide) to 3 day old maize seedlings enhances tolerance against the chilling stress [39]. Meanwhile, Pulido et al. found that nuclear localization of peroxiredoxin and thioredoxin prevents nucleic acids from oxidative damage occurring during the maturation and germination in wheat seeds [40]. The detoxification of H2O2 in the seed filling is catalyzed by the isoforms of catalase (CAT). The isoform CAT3 is involved highly in the early postpollination, whereas CAT1 and CAT2 isoforms play a crucial role during the seed development [41, 42]. Recently, Leymarie et al. clearly demonstrated the necessity of the ROS and the antioxidant enzymes for successful germination using mutant seeds. In the *Arabidopsis cat2-1* mutant, intracellular H2O2 and redox perturbation were increased. In case of the *vte1-1* mutant lacking a gene that encodes the tocopherol cyclase, an increase in the redox active biosynthetic intermediate was observed [29]. Tocopherol is generally involved in the protection of lipids from oxidation. Tocopherols, also called vitamin E, functions as terminators of a PUFA recyclable chain reaction. The tocopher‐ oxyl radical can be recycled back to the tocopherol by the reaction of ascorbate or other antioxidants [43]. The lack of the *vte1-1* function in the seeds releases lipid peroxy radicals. Although the *cat2-1* and *vte1-1* affect the seed germination to a certain level, plants lacking the *rbohD* gene can successfully complete germination (with time delay). The *rbohD* gene is involved in the conversion of the O2 and NADPH to form superoxide and plays a vital role in the cell-to-cell propagation of the ROS and generation of the •OH. The •OH is essential for

Nonenzymatic antioxidants that actively participate in the ROS equilibrium are ascorbate, glutathione, and preoxiredoxins [44–47]. Low moisture content decreases the molecular mobility and the accessibility of substrates for the catalysis of antioxidant enzymes [44]. Ascorbate plays a major role in the progression of cell cycle, cell growth, hormonal signaling pathways, and embryogenesis. Ascorbate content of the seed decreased the H2O2 by increasing

modifications, especially mRNA oxidations, are governed by the ABA [32].

80 New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology

**3.5. Antioxidant enzymes**

the cell wall loosening of endosperm [29].

**3.6. Nonenzymatic antioxidants**

It has been proven that an inhibitor of the ROS, sodium benzoate, decreases the germination rate of the seed [47]. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, also affects the germination rate [29]. On the other hand, methylviologen, involved in the release of superoxide from the mitochondrial respiratory chain breaks the seed dormancy [10]. Capacity of seeds to germinate or remain dormant is determined by the two important phytohormones such as GA (dormancy release) and/or ABA (dormancy induction). Bailly et al. reported that GA and ABA are interlinked with the ROS and the scavenging capacity of antioxidant enzymes [19]. Generally, GA is mainly used for the dormancy release, while ABA induces the dormancy. The GA is involved in the stimulation of •OH production, especially in the radicle, and it also downregulates the enzymes involved in the ROS detoxification. Contrastingly, ABA inhibits the Fenton reaction, where the iron (II) is oxidized by the H2O2 to form the iron (III) and the release of •OH [38]. The processes of seed germination and dormancy are linked with ROS accumulation [48]. The productions of H2O2 in the sunflower are higher in the germinating seeds than the dormant seeds [5]. Similar results have been observed by comparing the dormant and nondormant seeds of many plants, such as *Arabidopsis*[29], *Triticum aestivum* [49] and *Pisum sativum* L. [50]. Moreover, the H2O2 stimulates the signaling cascade which induces the expression of specific genes [24]. In addition to H2O2, accumulation of other ROS species such as O2 − and •OH has also been observed in various plant species [50–53]. Bazin et al. mentioned that the germination of sunflower seeds was associated with the mRNA oxidation. The oxidation level of mRNA was higher in dormant seeds as compared with the nondormant seeds [32]. Genes such as *GA3ox1* and *GA3ox2* are involved in the synthesis of active GA [54]. Ishibashi et al. observed the induction of H2O2 in the aleurone layer by the GA in *Hordeum vulgare*, whereas ABA suppresses the production of H2O2. Furthermore, exogenous addition of H2O2 degrades Slender1 (SLN1), a well-known repressor of GA. Due to the induction of αamylase (α-amy) and GAMyb, we can consider that H2O2 acts as an antagonist to ABA [55]. Cross-talk of the ROS with the phytohormones was reported to be mediated by the influx and efflux of Ca2+ ions [56, 57]. Bethke and Jones stated that H2O2 was involved in the programmed cell death [58]. Contrastingly, ABA increased the tolerance against the PCD [59]. Recent report suggested that the NO is also involved as a signaling messenger during the seed germination and dormancy process [60].

#### **3.8. Protection against pathogens**

The release of the ROS in the seeds during the development period protects the seeds against pathogens. It also induces the systemic-acquired resistance (SAR) and PCD. Especially when the ROS is mobile toward the seed coats, aleurone layers, and embryonic axis, the attack of microorganism is prevented by the induction of SAR and PCD [19, 61]. Briefly, the plasma membrane NADPH oxidase produces O2 − , which is converted into H2O2 by SOD during the infection. Subsequently, H2O2 induces a hypersensitive reaction which leads to PCD of the infected cell. Eventually, H2O2 can also directly affect the pathogens [62, 63]. The main categories of genes involved in the H2O2 induction are related to defense, transcription, signaling (e.g., phosphatases, kinases), and importantly ROS synthesis and degradation. Perturbation of endosperm for the radicle emergence leads to the induction of defense-related genes. It helps to protect the newly germinating seeds from the pathogens [64]. During the seed germination process, lower concentrations of the ROS are involved in the cell signaling process, whereas higher concentrations trigger the PCD to facilitate the radical protrusion [65].

#### **3.9. Endosperm weakening**

Proteolytic cleavage of cell wall polymers is induced by the hydrolases such as mannose, glucanase, and cellulose. The scission of polysaccharides is a vital step to determine the radicle emergence. According to Muller et al., •OH accumulation is the main factor, which influences endosperm weakening by the breakdown of H-bonds in the cell wall-polysaccharide required for the radicle protrusion. Generally, •OH is extremely reactive and is considered as the most aggressive form of oxygenated derivatives [26]. Uncontrolled ROS accumulation affects the

**Figure 1.** Schematic representation of the involvement of the reactive oxygen species (ROS) in the quiescent seed for the nondormancy and dormancy conditions. Dots represent the accumulation of ROS, blue for superoxide (O2 - ), brown for hydrogen peroxide (H2O2), and purple for hydroxyl radical (•OH). The LEA, late embryogenesis-abundant pro‐ teins; SAR, systemic acquired resistance; PCD, programmed cell death; LOO•, lipid peroxyl radical; LOOH, lipid hy‐ droperoxide.

integrity of DNA, causing changes at the sequence level that impair proper seed germination and could be able to change the genetic code of the seeds. The O2 − and H2O2 seem to be less reactive toward nucleic acid as compared to •OH [38]. However, cellular dysfunctions caused by ROS accumulation can be prevented by the antioxidants [40].

Oxidative damages caused by excess ROS are irreversible. The progressive conditional, temporal, and spatial distribution of the ROS tightly controlled by the antioxidant system leading to seed germination are also defined as 'oxidation window' (**Figure 1**).
