Reactive Oxygen Species (ROS): Beneficial Companions of Plants’ Developmental Processes

1Life on the earth began under a reducing atmosphere. About ∼2.7 billion years ago, the introduction of O2-evolving photosynthetic organisms led to an accumulation of O2 that changed the reducing environment into an oxidized one. Since then, reactive oxygen species (ROS) have been unavoidable companions of aerobic life. Electron transport systems (ETCs) generally produce ROS by virtue of O2 being a powerful electron acceptor. ROS such as superoxide radical, hydroxyl radical (•OH), hydrogen peroxide (H2O2) and singlet oxygen (1O2) are either the product of oxidation–reduction (redox) reactions, or activated derivatives of O2, continually generated in chloroplasts, mitochondria, peroxisomes, and glyoxysomes, and also in the cytosol, apoplast, nucleus, and, endomembrane systems. These ROS are highly reactive and toxic, causing oxidative damage to macromolecules such as lipids, proteins, and nucleic acids. In photosynthetic tissues (leaves), chloroplasts are the major sources of ROS generation in plants (;;; Figure 1; Table 1). Photosynthetic electron transfer chains of chloroplast produce high amounts of (through leakage of electrons from the acceptor side of photosystem II to O2, from Fe–S centers of photosystem I and reduced ferredoxin (Mehler reaction; ), while in non-photosynthetic tissues of plants (i.e., roots, meristems, or seeds) mitochondria are the biggest sources of ROS generation. In mitochondria, the key sources of ROS production are NADH dehydrogenase complexes I and III, and the ubiquinone pool where radicals are generated from the complexes as a by product of energy metabolism by the reduction state of ubiquinone pool. Other sources of ROS, mainly in non-photosynthetic tissues are NADPH oxidases, cell wall peroxidases, peroxisomes, and glyoxysomes. In glyoxysomes and peroxisomes, H2O2 is produced during fatty acid oxidation (by Acyl CoA oxidase), and photorespiration (by glycolate oxidase), respectively. In chloroplasts, during impairment of CO2 fixation, an increased activity of ribulose-1,5-bisphosphate carboxylase/oxygenase leads to the formation of glycolate, which moves to peroxisomes and leads to the formation of H2O2, via its oxidation in the presence of glycolate oxidase enzyme (Figure 1; Table 1). Cell wall-associated extracellular peroxidases and plasma membrane-bound NADPH oxidases (e.g., Nox, Rbohs or respiratory burst oxidase homologs) are the key enzymes that produce H2O2 and (usually rapidly dismutated to H2O2) in the apoplast. In Arabidopsis, 10 genes encoding respiratory burst oxidase homologs (Rbohs, i.e., RbohA-RbohJ) have been reported. Notably, in presence of redox-active metals such as Fe2+ and Cu+, H2O2 will give rise to highly reactive •OH (the most toxic oxidant in the ROS family) or via the Fenton or Haber–Weiss reactions, respectively.

2Parallel to the production of ROS, aerobic organisms have evolved sophisticated and well-outfitted antioxidant defense machinery. This machinery possesses highly efficient enzymatic [superoxide dismutase (SOD); ascorbate peroxidase (APX); catalase (CAT); monodehydroascorbate reductase (MDHAR); dehydroascorbate reductase (DHAR); glutathione reductase (GR); glutathione peroxidase (GPX); glutathione-S- transferase (GST); and guaiacol peroxidase (GOPX)] and non-enzymatic [ascorbic acid (ASA); glutathione (GSH); tocopherols, phenolic compounds, and non-protein amino acids] antioxidant defense systems to control over the cascades of uncontrolled oxidation to detoxify ROS in order to balance the cellular ROS level, as the maintenance of redox homeostasis is essential. However, disturbance in the equilibrium between ROS and antioxidant defense system creates a condition of oxidative stress.

3Although early research was focused on the toxic nature of ROS, the interest has shifted over the last decade toward their emerging role as signaling molecules in a broad range of physiological processes, such as growth and development, seed germination, programmed cell death (PCD), root growth, and gravitropism. A tight balance between ROS production and scavenging is necessary for the regulatory action of ROS. The uses of ROS as signaling molecules indicate that plants have evolved the ability to achieve a high degree of control over ROS toxicity. In this review, we have summarized studies from past decade that have improved our understanding about roles of ROS in signaling and the regulation of cellular processes in relation to plant growth and development.

4Coupled oxidation–reduction (redox) reactions in cells are the necessity of life. Various cellular signaling events are mainly based on redox reactions; therefore, it is probable that ROS are directly linked to the cellular redox metabolism. A redox regulatory network is found in each cell whose state is adjusted by ROS and these ROS regulate gene expression, translation, metabolism, and turnover. Accordingly, cell maintains the redox homeostasis by powerful and complex antioxidants such as ascorbate and glutathione and/or antioxidant enzyme systems. Ascorbate and glutathione are much more than simple antioxidants, as they consist of both oxidizing and reducing forms. The antioxidants are principally maintained in the reduced state. A powerful reductant not only favors ROS removal but can also promote ROS generation. Alterations in the equilibrium of reduced vs. oxidized forms of the antioxidants might be used as a sensor for changes in the environment, and changes in ROS levels which might affect the redox status of the cell. Increased levels of ROS may result into the oxidation of antioxidant systems which change the redox equilibrium of the cell. According to in order to allow appropriate retrograde signaling to the nucleus, coordination of gene expression in between the compartments of the cell needs monitoring of chloroplast, glyoxysome, mitochondrial and peroxisome status. These facts imply that cells have evolved strategies to operate ROS as biological signals that control various developmental programs. This explanation is based on the statement that a ROS can interact with a specific target molecule which perceives the elevated ROS concentration, and after that translates this information into a change of gene expression. These changes in transcriptional activity might be achieved by the oxidation of components of signaling pathways which consequently activate transcription factors (TFs) or directly by modifying a redox-sensitive TF. ROS effects on components of the mitogen activated protein kinases (MAPKs) cascade result in the indirect activation of TFs. In Arabidopsis, H2O2 activates Arabidopsis thaliana MPK6 (AtMPK6) and AtMAPK3 by the activity of MAPKKK Arabidopsis NPK1-RELATED PROTEIN KINASE1 (ANP1; ) and strongly induces A. thaliana NUCLEOTIDE DIPHOSPHATE KINASE2 (AtNDPK2; ). In a recent study, a link between the ROS-response ZAT12 zinc finger protein and iron regulation in cells was explored which suggests that the equilibrium between ROS and iron is crucial for the growth and development of plants preventing the formation of highly toxic •OH. According to ZAT12 interacts and suppresses the function of FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTIONFACTOR, a central regulator of iron deficiency responses. In response to elevated ROS, ZAT12 is up-regulated and suppresses iron uptake thereby preventing the risk of •OH formation. Another example of biological regulatory circuit was reported by who have identified a WRKY TF that is phosphorylated by MAPK and a W-box in the promoter region of Nicotiana tabacum RBOH, interconnecting the phosphorylation events of MAPK in response to pathogen recognition with the accumulation of RBOH protein. Moreover, in recent years different members of the NAC family of TFs (e.g.,;; ), APETALA2/ethylene response TF REDOX RESPONSIVE TRANSCRIPTION FACTOR1 that are regulated by different WRKYs, and different zinc-finger proteins such OXIDATIVE STRESS2 like ROS-response regulatory proteins were identified. Besides TFs, calcium (Ca) waves comprise important components of systemic signaling in plants (e.g.,;;; ). Several findings summarized that RBOH acts as a central hub in the cellular ROS-signaling network and this RBOH functions along with MAPK pathways to integrate ROS signals and modulate cell-to-cell signal propagation in local and systemic signaling. These MAPK pathways take part in retrograde signaling from the chloroplast to the nucleus. Thus, MAPK pathways are of fundamental as well as far-reaching importance in converting ROS signals into protein phosphorylation.

5In multicellular organisms, growth mainly depends on the maintenance of an appropriate equilibrium between cell division and differentiation (Figure 2). In the case of animals, interruption of this equilibrium between cell division and differentiation can lead to tumoral growth and disease, while in plants; it can lead to premature cessation of organogenesis, or as a consequence of abnormal growth. The initial stage of differentiation is marked by the transition from cellular proliferation to elongation, which is regulated by ROS homeostasis. According to, and H2O2 are two main ROS, which are differentially distributed within the root tissues of model plant Arabidopsis. principally accumulates in expanding meristem cells, while H2O2 accumulates in the elongation zone and an overlap of both types of ROS is observed within the “transition zone” (Figure 2). The transition between root cell proliferation and differentiation is mainly controlled by the delicate equilibrium between and H2O2, which in turn is regulated by a TF: UPBEAT1 (UPB1; ). The UPB1, a member of the basic/helix loop-helix (bHLH) TF family, shows an increased expression in the root transition zone. have reported that plants over-expressing UPB1 had shorter roots due to a decrease in both meristem size and mature cells, while plants lacking UPB1 (upb1 mutant) had longer roots with increased meristem size and longer root cells. Furthermore, when investigated the mechanism of action of UPB1 regulated genes, a set of peroxidases (Prxs) was seen to be directly repressed by UPB1. Further investigation pointed out that production was reduced in lines over-expressing UPB1 but increased in the elongation zone in the upb1 mutant, while H2O2 was found to be increased in the plants over-expressing UPB1 and reduced in the elongation zone of upb1 mutant. These findings suggested that the position of the transition zone is determined by the ramp formed by in the meristem to maintain the cellular proliferation, and H2O2 in the elongation zone, for differentiation. This indicates that UPB1, feedback of which is regulated by H2O2, plays a key role in maintaining the balance of and H2O2 via its control on Prxs expression.

6Genetic analysis by signifies that the subunits of the mediator complex PHYTOCHROME AND FLOWERING TIME1 (PFT1)/MED25 and MED8 are vital for the differentiation of root hairs (Figure 2). Moreover, studies have demonstrated that PFT1/MED25 restricts cell growth, while MED8 works independently of PFT1 to control organ growth and regulates ROS balance in roots. Transcriptional profiling of roots of pft1 mutants disclosed that PFT1 triggers H2O2 formation mediated by a subset of class III peroxidase enzymes, to maintain the equilibrium between H2O2 and during root hair differentiation. Perturbed H2O2 and distribution was seen in pft1 mutants, suggesting that PFT1 is necessary to maintain the redox homeostasis in roots.

7In the fungus Neurospora crassa, at the start of each cell differentiation step, a hyperoxidant state occurs that leads to the development of conidia. According to, the hyperoxidant state is transient and unstable, and occurs as ROS production exceeds the antioxidant capacity of the cell. It is well-known from this example that antioxidants inhibit developmental processes, which leads to the idea that ROS may be required for these processes in plants as well. In contrast, it is predictable that the deficiency of antioxidant enzymes will result in to elevated ROS levels, and improved cell differentiation will take place.

8Programmed cell death, which is critical in plant organogenesis and survival, is an integral cellular program employed by plants by which targeted cells terminate to demise under certain developmental (vegetative and reproductive) and stress conditions. It is essential for removing unwanted diseases or damaged cells, controlling cell number and maintaining the homeostatic balance and thereby improving the endurance of organisms.

9Plant PCD is related with a number of developmental processes including embryo formation, degeneration of the aleurone layer in germinating seeds, formation of root aerenchyma and epidermal trichomes, differentiation of tracheary elements, tapetum degeneration, pollen self-incompatibility, floral organ abscission, leaf shape remodeling, and leaf senescence. ROS such as H2O2 is identified as key modulators of PCD along with other biological processes such as growth and development. Concerning developmental programmed cell death (dPCD), a division can be made between (1) differentiation induced PCD that occurs as final differentiation step in specific cell types, for instance, tracheary elements in xylem, the trichome/ root cap, or tapetum layer of anther, and (2) age-induced PCD, i.e., cells must die to form organs for appropriate functions or shapes (unisexual reproductive organs in dicots, aerenchyma tissue, leaf shape), or cells die as they accomplished their function and/or are no longer required (nectaries and petals of flowers after pollination, leaf senescence; ). Nevertheless, some types of PCDs, including endosperm cell death or pollen self incompatibility (SI) does not fall into either of above proposed classes.

10The life cycle of plant alternates between diploid sporophyte and haploid gametophyte phases. The sporophytic phase starts with zygote (2n) formation and ends with flower bearing adult plant while gametophytic phase starts with sporophytic meiosis and develops into male (pollen grain) and female (ovule) reproductive structures. In dPCD, plants thereby removing tissue or organs which are no longer necessary conserve its energy along with blocking the possible entry sites for pathogens.

11According to, to protect the initial growth of the ovule, sepals, and petals after accomplishing their role may undergo dPCD, where ROS specifically H2O2 is known to be involved in the death of petal cells. ROS is produced from H2O2 therefore, the enzymes regulating H2O2 level showed differential expression in dPCD during senescence. have also reported an increase in ROS due to increased SOD and decreased CAT activities in daylily plant that results into dPCD.

12In megaspore, the chalazal cells differentiated into the three antipodal cells and micropylar cells develop into one egg and the two synergid cells and in center of embryo two polar nuclei are present. The synergid cells produce signals which attract and guide the pollen tube growth, ultimately deteriorate in a programmed manner. In A. thaliana, FERONIA (FER) receptor kinase is responsible for the production of ROS by NADPH oxidase via ROP small GTPase in synergid cells leading to dPCD. ROS by activating Ca2+-permeable channels alter the cell wall extensibility. In A. thaliana, ROS production (necessary for pollen tube growth) by the pollen-specific NADPH oxidases: RbohH and RbohJ is mediated by ANX, which maintains a tip-focused Ca2+ gradient. The excess Ca2+ influx might be the reason of ROS-induced pollen tube rupturation/PCD. Likewise, synergid-derived ZmES4, a defensin-like peptide by opening the K+ channel induced rapid pollen tube rupturation in maize. These findings revealed that ROS and other secreted proteins regulate cell wall alteration for the reception of pollen tube through the functions of FER in synergid cells and ANX in pollen tubes. On the opposite end of the ovule, three antipodal cells undergo dPCD immediately before fertilization in mature embryo sacs of A. thaliana. It is reported that lifespan of antipodal cell is regulated by central cell so; ROS accumulation specifically in the mitochondria of the central cell might act as a signal in dPCD of antipodal cell in a non-cell autonomous way.

13During development of male sex organs, ROS signatures dictate the correct timing of tapetal dPCD because in order to release pollen, tapetum cells must die. In tapetal dPCD, H2O2 acts as a key regulator. In A. thaliana RBOHE, a tissue-specific NADPH oxidase is the major H2O2 contributor supporting tapetal cell death. According to both overexpression and deficiency of RBOHE eliminated usual ROS accumulation leading to the male sterility.

14ROS also play a signaling role during self-incompatibility- (SI; to prevent inbreeding by the rejection of incompatible pollen) induced pollen PCD in Papaver. According to, the simultaneous scavenging of H2O2 and NO suppressed SI PCD. SI leads into signal transduction that involves increased ROS, Ca2+, NO, activation of MAPK, and protein phosphorylation as it is supported by large number of evidences that ROS play a key role in SI response that activates dPCD in self-compatible pollen. In poppy, SI is associated with ROS induced activation of Ca2+ signaling cascade followed by a release of mitochondrial cytochrome c into the cytosol and caspase-3-like enzyme activity, and process is mediated by the activity of MAPK cascade signaling (; Figure 1). According to, ) Ca2+ influx boosts H2O2 levels to SI-inducing levels that finally triggered a signaling network leading PCD thereby ensuring that fertilization is not achieved by incompatible pollen.

15During pollination, when stigma is receptive (ready to receive pollen grains), accumulates high H2O2 levels, and that H2O2 levels decrease when stigma starts to support pollen development. After that, the signal exchanges in between the stigma and the pollen, that might regulate the production of ROS and reactive nitrogen species (RNS) in both tissues takes place. The H2O2 level in the stigma which was increased before pollination was found decreased after pollen arrival. Contrary to this, the and NO levels were increased with attendant increase in peroxynitrite (ONOO-; ). Treatment with ONOO- scavengers decreases papillar cell death and also reduces the quantity of pollen grains deploying dPCD, suggesting that ROS mediated PCD signaling takes place during incompatible pollination in the olive. After enterance in female gametophyte, pollen tube discharges its content (two male gametes). Out of two male gametes, one fertilizes the binuclear central cell to form a triploid endosperm and other migrates to the egg cell. The endosperm functions as a storage tissue for developing embryo which after sometimes undergo ROS induced PCD mediated by abscisic acid (ABA) and ethylene.

16During cereal seed germination, gibberellic acid (GA) is produced by plant embryo that activates the aleurone cells to release an amylase enzyme, which sequentially hydrolyzes and mobilizes starch from the endosperm in the seeds thereby providing energy to the embryo. After completion of germination, these aleurone cells are abolished by PCD. According to, ROS and GA play vital roles in the control and implementation of aleurone PCD (discussed in next section).

17Besides embryonic development, ROS also play a key role in dPCD of vegetative parts; the development of xylem tracheary elements is one of them. Contrary to other cell type in plants, xylem tracheary elements are functionalized by PCD leading into hollow cell cadavers with merely a remaining cell wall demarcating the sap conducting cylinder. H2O2 by increasing cross-linking of polymers induces stiffening of the cell wall as growth ceases next to onset of differentiation. Lignification in stem of Zinnia elegans L. is characterized by a burst in H2O2 production, which could act as a developmental signal in secondary wall differentiation. This H2O2 may coordinate and regulate at the transcriptional level mRNAs synthesis encoding phenylalanine ammonia-lyase like lignin biosynthetic enzyme, and xylem peroxidases or trigger xylem differentiation thereby inducing PCD and formation of secondary cell wall. The major H2O2 production site in the differentiating xylem is outer-face of the plasma membrane of both non-lignifying xylem parenchyma cells and differentiating thin-walled xylem cells. From their sites of production, H2O2 diffuses (mainly through the continuous cell wall space) to the differentiating (cells which are at the state of secondary cell wall-formation) as well as differentiated xylem vessels (cells already have completed secondary cell wall formation, i.e., lignified; ), and H2O2 is thought to not limit the rate of xylem lignification. These findings strongly supported that H2O2, which is a key component for inducing dPCD signaling network in lignifications of xylem vessels, is mainly produced from non-lignifying xylem parenchyma cells as it is necessary for the polymerization of cinnamyl alcohols.

18A similar picture has been reported in various forms of developmental cell death— formation of aerenchyma and the senescence-associated cell death. According to, ROS like and H2O2 are the key coordinators of senescence. In the internodes of rice, the exogenous application of H2O2 promotes the formation of lysigenous aerenchyma. In cortical cells of maize roots, have reported a strong up-regulation of gene encoding RBOH, thereby suggesting that RBOH mediated ROS generation participates in the formation of lysigenous aerenchyma.

19In many plants, trichomes in their fully differentiated stage are dead. The development of trichome trails a switch from mitosis to endoreduplication, branching of cells, expansion, and ultimate cell death initiated by H2O2 burst. In trichomes of succinic semialdehyde dehydrogenase (key enzyme of γ-aminobutyrate metabolic pathway) knockout plants, enhanced H2O2 levels are observed. In the stem nodes of rice plants, the epidermal cells that cover the primordia of adventitious roots undergo cell death before the emergence of adventitious root. Ethylene promotes epidermal cell death, which is mediated by an excess H2O2 production. In rice plant, a non-enzymatic H2O2 scavenger metallothionein MT2b when downregulates it promotes ROS level in cell. In case of epidermal cells, ethylene downregulates MT2b that results into PCD thereby revealing that ethylene promotes ROS accumulation that lead to MT2b mediated cell death.

20Significant roles of ROS have also been reported during natural course of senescence, the last step of leaf development in the life span of an annual plant. The accumulation of ROS have been suggested to be an age-associated factor that triggers leaf senescence. proposed that ROS-triggered senescence is not caused by physicochemical damage of the cell, but that these molecules (ROS formation due to leakage of electrons from ETC to O2 at the onset of senescence) behave as signals, which activate gene expression pathways leading to suicide events. During cell senescence, lipid peroxidation can be activated either by lipoxygenases in some tissues, where the activity of lipoxygenase increases with increasing senescence, or by ROS. Therefore, it was concluded that, during senescence, lipoxygenase plays an essential role in promoting oxidative damage, in which not only stimulates lipid peroxidation but it can also form 1O2. have also reported that in senescent leaves, H2O2 content increases in parallel with an increase in lipid peroxidation and protein oxidation. So it is obvious that H2O2 plays a crucial role in the regulation of senescence signaling.

21After senescence, the resulting plant organ detaches from main plant body and the process is called abscission. In several studies, it has been reported that Prxs are regularly expressed during diverse types of abscission.

22Before discussing the role of ROS in leaf abscission, the notion should be recall that Prxs actively participate in the cell wall loosening. demonstrated that continuous H2O2 production in the cell wall participates in leaf abscission signaling, which is needed to induce the expression of cellulase enzyme (the cell wall-degrading), and when this continuous H2O2 production was suppressed by ROS inhibitors, it inhibited the cellulase activity and consequently inhibited abscission. On the other hand, exogenous application of H2O2 enhanced cellulase expression and abscission therefore, signifying that H2O2 production is essential to induce abscission. After continuous cellulase production (induced by H2O2), enhanced H2O2 generation at the Abscission-Zone in the Abscission-Zone-separating period was observed that might play a role in the cleavage of wall polysaccharides and loosen the cell wall. Therefore, a huge ROS production that probably driven by Prxs at the late stage of abscission may be associated with the cell wall degradation process.

23Seed dormancy and seed germination mechanisms are essential processes in plant development, and are believed to be a part of a complex regulatory network (Figure 3). In this network, ROS are considered to be key signaling actors as reported in A. thaliana, barley, cress, wheat, and sunflower. Seed germination starts with water uptake, along with initiation of cell division, and ends with radicle protrusion. In dry seeds, due to highly reduced enzyme activities, ROS are probably originated from lipid peroxidation like non-enzymatic reactions that occur even at very low moisture contents. However, in hydrated seeds (that occurs following imbibition) ROS may generate from all metabolically active compartments like chloroplasts (by election transfer in photosystems), glyoxysomes (by lipid catabolism), mitochondria (via respiratory activity), peroxisomes (by purine catabolism), and plasma membranes (by NADPH oxidase; ). In hydrated seeds, the continuation of respiration (in mitochondria) in imbibed seeds can lead to electron leakage because the electrons of mitochondrial electron-transfer chain (mETC) have sufficient free energy to directly reduce the O2, and this can be considered as unavoidable source of increased ROS production in mitochondria. Due to this reason, in hydrated seeds, the mitochondrial activity is considered as major source of ROS (such as H2O2) production during germination. Moreover, in hydrated seeds, ROS targets may be close or far from their production sites as the free water allows ROS (or their longer-living or movable forms like H2O2) to move and reach their targets farther from their production sites while, in dry seeds the ROS targets must be close to their production sites. It has been reported that H2O2 is produced in the seeds of Zinnia elegans, maize, wheat and soybean during the early imbibitional period, and cellular responses to H2O2, which is the most labile ROS messenger, mostly rely on the involvement of redox-active proteins, stimulation of redox-sensitive TFs that orchestrate downstream cascades, oxidation of specific peptides, and the activation of MAPKs. ROS produced during imbibition in different sub-cellular compartments, largely affect the expressions of various genes. The sub-cellular ROS interact with specific target molecules (particular for increased ROS concentration), which there translate that information by altering the gene expression. Oxidation of certain components that keep on to activate TFs of the signaling pathway, or the direct modification of redox-sensitive TFs by ROS, may led to the alteration of transcriptional activity.

24It has been reported in several studies that during seed imbibition, ROS oxidize specific proteins for driving the germination process, or oxidize mRNAs to prevent their translation.

25According to, ROS have well-recognized role in endosperm weakening, protection against pathogens, mobilization of seed reserves, and PCD in seeds. In some seeds, an endosperm covers the radical tip and acts as a mechanical barrier in radicle protrusion.

26When addressing the event of seed germination and seedling establishment, it appears exceedingly relevant to consider and investigate the possible interaction between ROS and plant hormones in the process of germination, as ROS involving PCD process have been established in aleurone layer of cereal grains, which are closely related to H2O2 interactions with GA and ABA hormones (;;; Figure 3). It is known that in aleurone cells of cereals, GA supports germination as well as post-germination processes, which are inhibited by ABA. Although GA downregulates the ROS scavenging enzyme activities, they are retained by ABA, which advocates a ROS link to PCD execution. In vivo, ROS can damage/oxidize/weaken the cell wall polysaccharides, activate calcium channels, Ca2+ binding proteins, MAPK cascades, and damaged cell wall polysaccharides, permit elongation of the growing radicle, which is a characteristic feature of endosperm rupturation in the aleurone layer.

27These authors showed that wall polysaccharides are oxidized in vivo by the developmentally regulated action of apoplastic •OH. The cell wall loosening is assumed to involve the enzymatic as well as non-enzymatic cleavage of structural polysaccharides. The regulated action of apoplastic •OH radical, which is generated by Prxs and/or the Fenton reaction in the cell wall (reaction 1) of radicles and endosperm caps, is known to cause the cleavage (breakdown) of hyaluronate, chitosan and pullulan-like polysaccharides.

28The production as well as the action of •OH increased during endosperm weakening and radicle elongation, and were inhibited by ABA. These outcomes were reversed by GA, representing a positive role of •OH in cell wall loosening during seed germination.

29When aleurone cells are exposed to GA, α-amylase and other hydrolytic enzymes are synthesized and secreted. Contrary to this, antioxidants (activities of CAT, APX, and SOD) suppressed the induction of amylases. While ABA acts as an antagonistic factor blocking the GA response in aleurone cells via the expression of a different set of genes- reinforcing the idea that H2O2 functions in GA and ABA signaling thereby regulating α-amylase production in aleurone cells.

30In seeds, a majority of studies have reported that ABA suppresses ROS generation, i.e., it acts as a negative regulator during seed germination while behaves as a positive regulator of the induction of dormancy. In presence of GA, exogenous H2O2 had minor effect on the degradation of primary transcriptional repressor, i.e., DELLA proteins Slender1 (SLN1) mediating GA signaling, but it supported the production of the mRNA encoding GAMyb (GA-regulated Myb TF) that acts in downstream of SLN1 protein and engages α-amylase mRNA induction. Moreover, H2O2 holdbacks the production of ABA-responsive protein kinase (PKABA) mRNA, which is induced by ABA; the production of GAMyb mRNA is repressed by PKABA. From these studies, have concluded that the repression of GAMyb mRNA was released by H2O2 via PKABA and this H2O2 subsequently promoted the production of α-amylase mRNA, thereby suggesting that GA generated H2O2, act as a signal that antagonizes ABA signaling in aleurone cells.

31Ethylene is another hormone that acts as a positive regulator of seed germination. In soybean seeds, observed that during seed imbibition ROS are produced, which further promotes ethylene production in sunflower seeds. Recently, have reported that the interaction between ABA and ethylene have antagonistic effects during seed germination. Moreover, by giving special consideration to cross-talk between ABA, ethylene, and ROS, mechanisms of action of ROS in the process of germination have been elucidated (Figure 3).

32According to ROS also participate in breaking seed dormancy and in germination by activating the oxidative pentose phosphate pathway (oxPPP). The authors observed that the exogenous application of H2O2 induces the carbonylation of enzymes taking part in restoration of reducing power during glycolysis, which stimulated oxPPP. Moreover, a relationship between H2O2 contents and gene expression related to MAPKs has also been noticed by. According to during seed germination, MAPK cascades affect cell division and the hormone so, pointed out that variations in H2O2 contents (increases during germination) of seed may induce germination thereby activating those MAPK cascades.

33After seed germination, the part of seedling which have to make root and shoot, sense the direction of the gravity vector and elicit gravitropic growth, generally sending the roots to downward and shoot to return to upward and directed growth. Generally it is accepted that growth responses reveal differential auxin redistribution to the flanks of the organ that results into reduction of elongation on one side and acceleration on the other. This differential growth in response to gravity leads to the bending of organ. These gravitropism responses have been connected to ROS generation in the roots of Arabidopsis and maize. In gravistimulated roots, ROS accumulation was found asymmetrical to the lower cortex within 30 min of reorientation, which becomes symmetrical upon longer stimulation. Asymmetrical application of H2O2 impregnated agar block causes root bending toward block. Here, some caution must be exercised such as H2O2 application will also interact with the wall that might have profound effect on cell extensibility. ROS are well-known to play a critical role in cell integrity that might also potentially lead to growth arrest. However, treatment with N-acetyl cysteine (NAC)-like ROS scavengers was observed to inhibit the response or curvature without affecting growth rate thereby suggesting that asymmetrical ROS generation is responsible for the gravitropic response. As the gravitropic responses are known to originate from inhibition of root elongation on the lower flank of the root, these findings are reliable with ROS accumulation to the lower flank acting as a growth inhibitor.

34It was thought that auxin might be a primary regulator of such kind of root elongation where it is accumulated on the lower flank to inhibit the growth of gravistimulated root. Consistent with these thoughts, reported that auxin can evoke ROS generation in a process which shows a close parallels to the animal model of ROS generation via NAPDH oxidases. A similar story is also emerging of a potential role of ROS in the gravitropic response of the maize roots. The role of ROS in auxin-induced root gravitropic responses has been reported in Zea mays, and results strongly suggest that ROS-mediated auxin redistribution by gravity stimulates an increase in gravitropic curving. Firstly, noticed that ROS are formed subsequent to gravitropic stimulus. When maize roots were kept horizontally, the ROS generation was detected in the apex, stimulating the gravitropic response. Secondly, ROS production is increased by auxin application to root cells in intact plants as well as in root protoplast cultures. Further treatment of roots with N-1-naphthylphthalamic acid (NPA, an auxin transport inhibitor) results in inhibition of gravitropism and the effect of NPA can be reversed by adding H2O2 which has indicated a direct causal relationship between auxin, ROS, and gravitropism. It has been observed that root bending, after pretreatment with the auxin transport inhibitor NPA, was brought about by unilateral ROS application to vertical roots. However, ROS scavenging by the antioxidants NAC, trolox and ASA inhibits root gravitropism. However, the effect of NPA was reversed by addition of H2O2. pointed out that ROS might work by kinase activation, but also considered that other mediators of the gravitropic response, such as inositol (1,4,5)-trisphosphate and calcium, might be involved. Furthermore, observed that phosphatidylinositol 3-kinase is involved in auxin-mediated ROS production that regulates root gravitropism while retreatment with the phosphatidylinositol 3-kinase activity inhibitor LY294002 blocked the auxin- mediated ROS generation, decreasing the sensitivity of root tissue to gravistimulation.

35It is well-known that in plant gravitropism, redistribution of auxin plays an important role in ROS mediated root gravitropism. To find out the role of auxin and ROS, used the auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA) and naphthylphthalamic acid (NPA) and a secondary metabolite coumarin which are known to alter the gravitropic responses in the roots of A. thaliana. Coumarin itself did not show any change in gravitropic response, but when added with NPA or TIBA, it reversed the effect of inhibitors. Moreover, studied ROS distribution in root tips and reported that NPA or TIBA causes distribution of around the root tip, which disappeared after coumarin addition to both treatments, restoring ROS localized distribution. These findings suggest that coumarin effect in restoring the root curvature did not depend upon auxin redistribution, but was mediated by ROS generation. Further to investigate the localization of ROS, by using blue formazan (NBT; precipitates accumulation) and 3,3′-diaminobenzidine (DAB; for H2O2 accumulation) demonstrated that was localized within the tip of root primordia, vascular cylinder cells as well as in the distal and middle parts of newly formed organs during the early stages of rhizogenesis; while H2O2 was pronounced in cortical and vascular bundle cells. On adding DPI to the medium, accumulation was then restricted to epidermis cells, while that of H2O2 was limited in vascular tissues. These findings suggest that engages itself in the process of rhizogenesis, while H2O2 is engaged in developmental processes such as cell growth.

36In plant system, the tip growth in either of the cases is mainly associated with the deposition of membrane and new wall materials centered toward the apex of elongating cells, thereby resulting into the formation of tube-like structure. To produce a correct shape and size, cell expansion needs to be carefully regulated. Polar cell growth is continued by oscillatory feedback loops including three main components, i.e., ROS, Ca2+ and pH that together play an important role in regulating this process over time. Apoplastic ROS balance controlled by NADPH oxidases and class III peroxidases has a great impact on cell wall properties during cell expansion.

37During expansion of polar cell (root or pollen), the apical zone is characterized by a tip-focused elevated cytoplasmic Ca2+ (cytCa2+) gradient and related proteins along with the apoplastic ROS (apoROS) generation. Cell takes Ca2+ either from external sources or stored in the cell wall and this Ca2+ released by changing apopH which is mainly controlled by plasma membrane located activation/deactivation of H+ pumps (AHA). Additionally, Ca2+ can be stored in vacuoles and ER-Golgi and released into the cytosol. To maintain low concentrations of cytCa2+, autoinhibitory P-type IIB Ca2+-ATPases (ACAs) transport Ca2+ back to the apoplast. In addition to this, the H+/Ca2+ antiporter activity of calcium-exchanger (CAX) translocates Ca2+ back to the apoplast and, at the same time, imports H+ into the cytoplasm. In the tip zone, high levels/concentration of cytCa2+ activates apoROS generation by NADPH oxidases (NOXs). Moreover, high levels of ROS quickly elevate the concentration of cytCa2+ by an unknown phenomenon. NOXH/NOXJ and NOXC were previously proposed to connect apoROS production with the rapid activation of Ca2+ channels (CaCs) of plasma membrane in growing pollen tubes and root hairs, respectively. The idea that ROS are important for sustaining polar growth came into existence with the cloning of root hair defective 2 (rhd2) mutants of A. thaliana. In rhd2 mutant background, according to and root hairs correctly initiated their developmental program and form bulges on the epidermal cell side but they fail to elongate/transition to tip growth. When alleles of the rhd2 were cloned, they were found to inhabiting a gene encoding the respiratory burst oxidase homolog C (AtRBOHC) of Arabidopsis, an NADPH oxidase similar to mammalian gp91phox (The phox91 family is conserved throughout the animal, plant and, fungal kingdoms; ), responsible for ROS production during oxidative burst. It means that RHD2 plays an important role in electron transfer from NADPH to an electron acceptor, and resulting in ROS formation. Besides RHD2, another target of ROS signaling in growing root hair has been reported, i.e., OXIDATIVE BURST INDUCIBLE1 (OXI1) kinase. OXI1 is a Ser-Thr kinase, which plays a role in elongation of root hairs, as it is slightly shorter in oxi1 mutant than wild type. It was suggested that RHD2-mediated ROS leads to the activation of a MAPKs cascade through OXI1 during growth of root hairs.

38A ROS burst is crucial for the rupturation of pollen tube and sperm release. Oscillatory growth is also related to changes in pH which is regulated by cation (H+)/anion (OH-)-permeable channels, by membrane H+-ATPases (AHA; ) and antiporters (Ca2+/H+ exchangers). AHA is supposed to be responsible for pumping H+ out of pollen tubes remains to be identified while AHA2 is highly expressed in growing root hairs. AHA directly controls apoplastic pH (apopH), which affects the enzymatic mechanism that alters cell wall components during cell expansion. Polar growth entails the stretching and deformation of the existing wall in the apical zone, which is escorted by the secretion of new wall materials. Oscillations in Ca2+, ROS and pH are coupled to transient cell wall loosening to facilitate turgor-driven localized cell expansion. In cells, growth is determined by the balance between cell wall loosening and stiffening. Root hair growth has to occur not only in accordance with the loosening of the cell wall to allow the expansion at its tip, but also with a need to avoid tip bursting by maintenance of wall integrity. In order to attain this pattern, the factors engaged in pollen tube growth such as cytCa2+ levels, pH, and NADPH are found to oscillate in such a manner in which the highest concentration follows the highest peak in growth, in concert to maintain the periodic oscillations. In root hairs, the maximum of these oscillatory fluctuations in apoROS concentration and apoplastic/cytoplasmic pH lead cell growth peaks by 7–8 s, while cytCa2+ oscillations delay oscillations in cell growth by approximately 5–6 s. In pollen tubes, the oscillations in cytCa2+ concentration are lagged by approximately 11 s in relation to cell expansion rates. Thus, polar cell growth is improved, and perhaps rapidly repressed, by high concentration of cytCa2+ and, consequently, high apoROS concentrations and a more alkaline apopH.

39In Arabidopsis, by using high-resolution growth measure-ments, showed that the frequency of growth pulses was approximately 3 per minute, similar to the frequency of pulses of ROS production and to the oscillations in the apical Ca2+ gradient. A comprehensive study of these oscillations implied a growth burst at 5 s due to an increase in tip-focused Ca2+ that may be mediated by a stretch-activated Ca2+ channel, which is followed 2 s later by ROS production in the wall. According to, this ROS production may play a role in covalent cross linking of polymers, and therefore strengthen the wall to prevent expansion and bursting. reported that tip growth ceased after the addition of external H2O2; however, scavenging of ROS promoted tip bursting, which suggests that ROS also play a role in wall rigidification. From these facts, it can be concluded that ROS-activated Ca2+-permeable channels induce Ca2+ influx to increase the cytCa2+ after the beginning of Rboh-mediated ROS generation, which further activates Rbohs. Therefore, Ca2+ increases in the cytoplasm probably response to increase ROS that alter Ca2+ influx.

40If we are talking about ROS signaling, it is noteworthy that changes in extracellular pH correlate with the oscillations in growth, which permit the alteration of tip growth in root hairs of Arabidopsis. The stiffening of the cell wall is carried out by both ROS and increased pH, which make cell wall resistant to turgor pressure. In the growing tip, oscillation of Ca2+ gradients is followed by alkalinization peaks in the apoplast along with the constitutive generation across the subapical part of the tip. An oscillatory component of extracellular pH at the tip of pollen tubes has been reported by, that changes the phase by producing ROS and increases the growth by increasing the Ca2+ accumulation. A similar series of events ROS, pH, Ca2+, and growth have also been reported in root hair tips. These findings put forward that one factor (either ROS or wall pH) compensate with at wall dynamics as well as in controlling the cytosolic activities needed to maintain the growth (Figure 4).

41Lateral root development is controlled by the antagonistic action of auxin and ABA. Auxin promotes the separation of pericycle initials and cell expansion while, ABA is essential during each stage of lateral root growth as it regulates the balance between cell proliferation and differentiation in the root meristem and in lateral root primordia. Both hormones also mediate ROS accumulation in growing roots and promote cell expansion. A transcriptomic analysis of roots treated with auxin revealed that majority of the peroxidase genes in the cs-SKP2B (SKP2B is a marker for lateral root development while cs-SKP2B dataset is its specific expression pattern of cell sorting that regulates the developmental program endogenously) dataset are not regulated by ABA. proposed that for growth regulation at root meristem, Prxs activity (regulated by UPB1) acts independently of the auxin pathway. In contrast, advocated that the auxin response involves ROS signaling. Peroxidases take part in lignin formation in the primary cell wall. It is notable that indole-3 acetic acid (IAA) is easily degraded by plant Prxs, which leads to the reduction of auxin pools. In tobacco plant, free IAA levels decreased in roots, indicating that alteration of auxin level is critical for the inhibition of lateral root formation in plants. Recently, it was proposed that root architecture is determined by the redox-mediated GPX family. Moreover,, through genetic analysis, showed that gpx mutants differentially affect lateral root formation (except gpx3-2 mutants), with all gpx mutants showing expanded lateral root primordia (LRP). have also observed that GPX1 and GPX7 are the two main Prxs to play a significant role in the regulation of lateral root development and root architecture, which is also associated with auxin-dependent control of lateral root formation. In addition, have shown that H2O2 and actively participate in lateral root development. They have also reported that in the cs-SKP2B dataset, several enzymes are involved in ROS formation, including AtrbohC, cytochrome P450 electron carrier proteins, and lipoxygenases. These findings suggested that ROS signaling is an important part of lateral root development. It can be summarized that Prxs activities (regulated by UPB1) regulate emergence of lateral roots via ROS signaling, most probably by promoting transition from cellular proliferation to differentiation (as discussed in previous section; Figure 2).

42The complex coordination between cell proliferation and cell expansion decides the final size of a single organ or organism. Plant leaves are initiated by the proliferation of meristematic cells and then by cell expansion without additional division in a second phase. An equilibrium between negative and positive regulators controls both phases by a large number of genetic pathways. For instance, TFs play a vital role. Of course, cell expansion is affected by changes in cell wall architecture and content. Such alterations may be due to Prxs that alter ROS levels in leaves.

43The Prxs particularly apoplastic, directly control stiffness of the cell wall, either by promoting or by restricting cellular extension. In the first scenario, generated by cell wall peroxidases, promote expansion by cleaving the cell wall polysaccharides and act as cell wall loosening agents. In contrast, H2O2 production in the cell wall promotes rigid cross-linking of cell wall components and results in growth restriction or making the cell wall stiffer. The activity and expression of Prxs are suppressed by KUODA1 (KUA1), a MYB-like TF that was found to function as a positive regulator of cell expansion during leaf development by altering apoplastic ROS homeostasis in A. thaliana. The overexpression of KUA1 results in increased cell size with larger leaves without affecting the cell number. In contrast, the kua1-1mutant has smaller leaves than the wild type due to a decrease in cell size, while the cell number was again unaffected. Moreover, the kua1 mutant shows elevated levels of H2O2 and increased activity of class III Prxs. The disturbance of KUA1 causes an increase in Prxs activity and results in smaller leaf cells. Therefore, expansion of the cell as well as the final size of the organ is controlled by ROS homeostasis as modulated by KUA1. This positive regulation/promotion can be interlinked with change in apoplastic H2O2 levels and it is noteworthy that changes in H2O2 level can also affect the pool. Of note, in the case of KUA1, H2O2 mediated inhibitory effect of Prxs, seems to be the fundamental link for the regulation of leaf cell expansion. The equilibrium between and H2O2 regulates cell proliferation and differentiation zones that decide the size of an organ (as discussed in previous section). showed that suppression of Prxs expression by KUA1 increased leaf cell expansion, without causing the increase in leaf cell proliferation. Moreover, the size of the first cortical root cell was decreased by H2O2 treatments, the root-localized Prxs are most probably essential to uphold low H2O2 levels to support root cell expansion. Contrary to this, also reported that inhibition of Prxs activity improves leaf size thereby suggesting that apoplastic Prxs mainly produce H2O2 that leads to cell wall crosslinking in leaves. Therefore, the impact of Prxs specifically their effect on the H2O2 level seems to comprise greater opposing effects on leaf growth in plants.

44In spite of amazing development in our understanding of ROS biology, the exact nature of the ROS-signaling network largely remains mysterious. The present review is an attempt to adress the regulatory action of ROS in plant growth and development. Earlier these were considered to be toxic by-products only, but now they have been found to function as central players in complex signaling networks. We have tried here to uncover the beneficial role of ROS as signaling molecules. Undoubtedly, in the past decade, our understanding in the field of ROS signaling have significantly improved but miles we have to go. We are entering in an exciting era of ROS signaling in plants. Our stage is set and it’s time to fit the different parts of the puzzle into its right place. Definitely, by uncovering novel biological roles of ROS, our understanding in the field of ROS signaling have become more than superficial, but many questions remain to be answered.

45For instance, is it possible to transport ROS from one subcellular site to another, and if possible, how do ROS signals journey within or across different cells?

46How are ROS-modulated gene networks linked with other signaling networks?

47Are they affected by changes in membrane potential?

48How does the effectiveness of ROS signals differ among sub-cellular compartments? How do NADPH oxidases participate in generating these signals?

49What are the concentrations of ROS in different sub-cellular compartments?

50Does each source contribute equally to the cellular ROS pool? Are specific ROS signatures induced by different stimuli?

51How do plant cells sense ROS?

52What are threshold levels of ROS at which they function as signaling molecules?

53Answers to these questions are crucial for elucidating mechanisms of ROS signaling. Our current understating regarding the involvement of ROS in developmental processes largely depends on some discovered components of signaling pathways such as NOXs, OXI1, Ca2+ influx and efflux, and some TFs. Further studies are needed to properly explain the complex regulatory machinery that integrates ROS signals with components of signaling pathways for the regulation of growth and development of plants. ROS are associated with many biotic and abiotic responses, and interpreting ROS signaling is expected to have a noteworthy impact on biotechnology and agriculture, possibly leading to crop development by enhancing yield under suboptimal conditions. Future work will no doubt reveal novel signaling roles for ROS and their interaction with other signals, and hence future of ROS research is very promising.

54VS and SP have conceptualized the review article. RS, SS, PP, RM, DT, VS, DC, and SP are being involved in writing this review article. RS and SS equally contributed in this review article.

55The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.