Linking Superoxide Production and Scavenging in Plant Development

ABSTRACT

Due to their strong oxidizing potential, rapid membrane permeability, and high reactivity, reactive oxygen species (ROS) play essential roles in plant development and stress responses. Superoxide (O₂ ^•‐) is a primary product of molecular oxygen reduction and a crucial source of hydrogen peroxide, representing a ROS species of substantial importance. Its detoxification is mediated by superoxide dismutases (SODs) and non‐enzymatic antioxidants such as ascorbate and tocopherol. The inherently unstable and dynamic nature of O₂ ^•‐ demands tight spatial and temporal control to preserve its signaling and developmental functions. The final O₂ ^•‐ level and its distribution result from the combinatorial effects of its production and scavenging, which is often mediated by phytohormones such as abscisic acid and auxin. The primary objective of this article is to elucidate the putative mechanisms underlying the coregulation of O₂ ^•‐ production and decomposition during plant development. We summarize current insights into the ABA and auxin‐mediated regulation of its production by NADPH oxidases and highlight the central role of SODs, enzymes responsible for O₂ ^•‐ detoxification, exploring also their key regulatory mechanisms. Using bioinformatics, we propose potential pathways coordinating O₂ ^•‐ production and scavenging. Mechanisms such as direct activation of SODs by ROS, transcriptional control, and protein‐protein interactions that respond to developmental signals are discussed.

Summary statement

This review examines phytohormone‐dependent regulation of NADPH oxidases and superoxide dismutases, controlling superoxide signaling in plant development. Superoxide production and detoxification are coordinated via posttranslational and transcriptional mechanisms and scaffold proteins.

1. Introduction

Reactive oxygen species (ROS) are metabolic byproducts generated by the excitation or reduction of molecular oxygen. Considering their oxidising properties, ROS possess major regulatory roles in various developmental processes and responses to different stimuli by affecting signalling pathways or regulating gene expression. Their levels may rapidly increase in response to environmental or endogenous stimuli, eventually leading to programmed cell death (PCD; Wang et al. ²⁰²⁴). The ROS production is compartmentalised to chloroplasts, mitochondria, plasma membrane, endoplasmic reticulum, cytosol, peroxisomes and apoplast. ROS derived from all sites has an indispensable function in various developmental processes (Kerchev et al. 2016; Considine and Foyer ²⁰²¹). Among the biologically relevant ROS (superoxide O₂ ^•⁻, hydrogen peroxide H₂O₂, singlet oxygen ¹O₂, and hydroxyl radical ·OH), O₂ ^•⁻ holds a unique significance because it is the primary product of the monovalent reduction of molecular oxygen. Moreover, it is an important source of H₂O₂ as it is a product of its dismutation, as well as ·OH, generated by the Haber‐Weiss reaction (Karpinska and Foyer 2024). O₂ ^•⁻ is believed to be unable to cross membranes, has a half‐life of less than 4 µs, and its migration distance is approximately 30 nm (Mittler 2017). Levels of O₂ ^•⁻ result from the combined effects of its production and scavenging mechanisms. The main sites of O₂ ^•⁻ production involve the photosynthetic and mitochondrial electron transport chains, plasma membrane‐localised NADPH oxidases (in plants encoded by RESPIRATORY BURST OXIDASE HOMOLOG genes, RBOHs), and cytosolic oxidases and dehydrogenases. Compartmentalised to these production sites, enzymatic and non‐enzymatic antioxidant systems have evolved to decompose O₂ ^•‐.

In addition, ROS, including O₂ ^•‐, closely cooperate with phytohormones such as auxin and abscisic acid (ABA) in developmental processes (Singh et al. 2024). Auxin controls diverse aspects of plant growth and development by regulating the fundamental cellular processes of expansion, division and differentiation, including the establishment of bilateral symmetry in an embryo, root hair development, root and lateral root development, shoot elongation, apical dormancy, gravitropism and phototropism (Du et al. 2020; Gomes and Scortecci ²⁰²¹; Roychoudhry and Kepinski ²⁰²²). ABA is an essential repressor of seed germination (Sajeev et al. 2024) and also plays a crucial role in regulating post‐germination growth arrest, particularly in response to adverse environmental conditions (Yadukrishnan et al. 2020). Furthermore, ABA is considered a floral suppressor and exhibits inhibitory roles in a floral transition (Martignago et al. 2020).

Little is known about the signals interconnecting O₂ ^•‐ production and decomposition during plant development. This review article provides a brief overview of the mechanisms by which O₂ ^•‐ regulates plant development, with a focus on auxin and ABA signaling. We summarize the current understanding of the regulation of its production mediated by RBOHs. The developmental roles of superoxide dismutases (SODs), the principal enzymes that decompose O₂ ^•‐, and their regulation are described in parallel. Finally, signals shared for both systems are delineated. We suggest possible coordination mechanisms of ROS production and antioxidant defense during developmental processes.

2. Superoxide‐Dependent Mechanisms Regulating Plant Development

Proper plant growth under optimal and stress conditions relies on the precise regulation of the cell cycle, proliferation, expansion, and polarity, processes tightly controlled by ROS, particularly O₂ ^•‐ and concomitant redox‐dependent mechanisms (Tsukagoshi 2012; Diaz‐Vivancos et al. ²⁰¹⁵; de Simone et al. ²⁰¹⁷). The multifaceted roles of O₂ ^•‐ in plant development have been extensively reviewed (Kim et al. 2007; Tsukagoshi ²⁰¹⁶; Lee et al. ²⁰¹⁷; Mhamdi and Van Breusegem ²⁰¹⁸; Foyer ²⁰¹⁸; Martin et al. ²⁰²²; Karpinska and Foyer ²⁰²⁴) and are not discussed here in detail; this section instead focuses on the mechanisms by which O₂ ^•‐ controls development.

Development is influenced by the quantity of O₂ ^•‐ produced and scavenged, as well as by its spatial distribution and localisation within cells and tissues. Moreover, as O₂ ^•‐ and H₂O₂ exhibit antagonistic effects and distinct tissue‐specific patterns, particularly in dividing versus differentiating cells (Dunand et al. 2007), developmental programs also depend on the concentration ratio of O₂ ^•‐ to H₂O₂. Synchronised spatiotemporal distribution of O₂ ^•‐ and H₂O₂ is required for PCD in tapetum during pollen development (Xie et al. 2014; Yi et al. ²⁰¹⁶), lateral root cap (Fendrych et al. 2014), or endosperm development (Young and Gallie 2000).

In general, ROS modulate the cell cycle, cell growth, differentiation, cell fate determination, and overall plant development primarily through oxidative modifications of key regulatory proteins, including transcription factors (TF), receptors, proteins involved in phytohormone signalling and metabolism, cytoskeletal constituents and cytoskeleton binding proteins (Livanos et al. 2012; Yang et al. ²⁰¹⁴; Tian et al. ²⁰¹⁸; Fichman et al. ²⁰²²; Figure 1). Due to the limited oxidation capacity of O₂ ^•‐, its impact on oxidative modifications is indirect, serving as a precursor of H₂O₂ and ·OH (Hino et al. 2024; Figure 1). For example, RBOH‐mediated O₂ ^•‐/H₂O₂ production is crucial for IAA3 oxidation during xerobranching (Roy et al. 2025). H₂O₂ generated from O₂ ^•‐ can also alter the biophysical characteristics of cellular membranes by oxidising membrane phospholipids, thereby impacting membrane fluidity and function (Parra‐Ortiz et al. ²⁰¹⁹).

Figure 1.

Schematic overview of the mechanism of ROS‐mediated plant growth regulation. ROS, such as superoxide (O₂ ^•⁻), hydrogen peroxide (H₂O₂) or hydroxyl radical (·OH), may affect plant development by multiple mechanisms listed in the boxes highlighted in green. These result in diverse molecular and structural outcomes noted in yellow‐highlighted boxes. The molecular and structural changes determine core cell developmental processes, which collectively drive plant growth and development.

Certain environmental conditions, such as high light, lead to the accumulation of O₂ ^•‐, which may target Fe‐S cluster‐containing proteins (Figure 1). Although not likely to occur in response to developmental cues, the overaccumulation of O₂ ^•‐ may hypothetically have developmental consequences by attacking the Fe‐S cluster‐containing proteins. Some of them are indispensable for mitochondrial respiration or chloroplast ETC, processes closely linked with development (Welchen et al. 2021).

O₂ ^• participates in the generation of ·OH, which is particularly potent in modulating cell wall properties by promoting wall loosening or stiffening, thereby influencing cell expansion (Schopfer et al. 2001; Kärkönen and Kuchitsu ²⁰¹⁵; Schmidt et al. ²⁰¹⁶; Figure 1). Lipid peroxidation, associated with increased O₂ ^•‐ dismutation by leaf FeSOD activity, is associated with floral transition in Arabidopsis thaliana (Ye et al. 2000). Unfortunately, the identity of the FeSOD isoform is unknown.

Plant development is further regulated through the interaction between ROS and phytohormones (Xia et al. 2015; Zwack et al. ²⁰¹⁶; Mangano et al. ²⁰¹⁷; Pasternak et al. ²⁰²³; Singh et al. ²⁰²⁴). ROS can influence auxin levels, disrupt its polar transport and distribution, or affect auxin signaling (Parveen et al. 2022). In this respect, numerous functions are mainly assigned to H₂O₂. In fact, the localization of H₂O₂, but not O₂ ^•‐, showed a polarized pattern in flax hypocotyl segments reprogrammed by exogenous auxin for the formation of adventitious roots (Takáč et al. 2016). However, O₂ ^•‐ is also linked to auxin signaling. Reduced O₂ ^•‐ production are associated with reduced auxin levels in the quiescent center and columella region, manifesting in shorter and thicker primary roots (Pasternak et al. 2023).

The formation of O₂ ^•‐ is also linked to ABA‐regulated primary root growth. The reduced O₂ ^•‐ production in rbohe and rbohd mutants alleviates the ABA‐mediated inhibition of primary root elongation in Arabidopsis (Jiao et al. 2013). ABA may also trigger mitochondrial O₂ ^•‐ production via mitochondrial DEXH RNA helicases ABA‐OVERLY SENSITIVE 6 (ABO6) and ABO8, in order to inhibit primary root growth by regulating root meristem activity and also seed germination (He et al. 2012; Yang et al. ²⁰¹⁴).

In addition, ROS may be directly involved in the metabolism of auxin and ABA. The RBOH‐generated O₂ ^•‐/H₂O₂ may have an impact on endogenous ABA levels, which occurs upon transcriptional regulation of 9‐cis‐EPOXYCAROTENOID DIOXYGENASE 1 (HvNCED1) and ABA 8'‐HYDROXYLASE (HvABA8'OH‐1) in barley (Hordeum vulgare) embryos (Ishibashi et al. 2015). They may regulate ABA catabolism (Liu et al. 2010), and they may directly oxidize IAA (Peer et al. 2013; Jenness et al. ²⁰²⁵). Nevertheless, a direct link between O₂ ^•‐ and auxin or ABA metabolism has not been reported to date.

3. Regulation of O₂•‐ Production by RBOHs During Developmental Processes

Plasma membrane RBOHs catalyze the generation of O₂ ^•‐ in the apoplast through electron transfer from NADPH to O₂. Subsequently, it is dismutated to H₂O₂ ether enzymatically by SODs (Wang et al. 2024), by antioxidants such as tocopherol and ascorbate, or by spontaneous dismutation, which is effective only at low pH values (3–6; with maximum rate at pH 4.8, corresponding to its pKa) and strongly depends on the concentration of O₂ ^•‐ (Sheng et al. 2014).

The Arabidopsis genome contains ten RBOHs, referred to as AtRBOHA‐J (Marino et al. 2012), which are distinctly expressed in various organs, tissues, and developmental stages, primarily fulfilling their roles through the production of O₂ ^•‐ and its subsequent dismutation to H₂O₂ (Chapman et al. 2019; Table 1). O₂ ^•‐/H₂O₂ produced by RBOHs modulate development through diverse mechanisms, including PCD, triggering of gene expression (Kai et al. 2016), alteration of cell wall rigidity (Kärkönen and Kuchitsu 2015; Orman‐Ligeza et al. ²⁰¹⁶), modification of hormonal signalling, and affecting lignin deposition (Hu et al. 2020).

Table 1.

An overview of the developmental roles of plant respiratory burst oxidase homolog (RBOH) genes.

Gene name (plant species)	Developmental process	Reactive oxygen species	Evidence	Citation
Pv PvRBOHA (Phaseolus vulgaris)	Lateral root initiation, emergence, and development	Unknown	Increased expression during lateral root formation	Arthikala and Quinto (2018)
			Increased signal of proRBOHA:GUS in emerging and developing lateral roots
			Reduced lateral root density in the RNAi line
AtRBOHA (A. thaliana)	Lateral root formation	H2O2	Expression in the basal meristem, where lateral root priming occurs	Orman‐Ligeza et al. (2016)
PvRBOHB (Phaseolus vulgaris)	Lateral root elongation	O2 •‐	Reduced O2 •‐ concentration in the apical region of the lateral root in the RNAi line	Montiel et al. (2013)
			Increased promoter activity during lateral root formation
AtRBOHB (A. thaliana)	Germination, radicle elongation	O2 •‐	Mutant shows delayed gemination and reduced protein oxidation	Müller et al. (2009)
AtRBOHC/RHD2 (A. thaliana)	Root hair formation	O2 •‐	Mutant has shorter root hairs and reduced O2 •‐ production in the root hair cells	Foreman et al. (2003)
				Orman‐Ligeza et al. (2016)
				Bhosale et al. (2018)
OsRBOH2, OsRBOH3, OsRBOH5 (Oryza sativa)	Root hair initiation and elongation	O2 •‐	Significant expression in root hairs	Wang et al. (2013)
				Kim et al. (2019)
OsRBOH8, (OsRBOHH) (Oryza sativa)	Adventitious root formation	H2O2	Detected OsRBOHH‐mediated ROS production	Li et al. (2025)
			Higher expression in the stem nodes and lysigenous aerenchyma
AtRBOHD (A. thaliana)	Germination	H2O2	The mutant shows delayed germination	Leymarie et al. (2012)
	Positively regulates primary root growth	H2O2	Increased expression in wild type roots in response to ABA	Jiao et al. (2013)
			Mutant shows the increase in H2O2 levels in roots after ABA treatments, and a shorter primary root
	Negatively regulates lateral root formation	O2 •‐	Mutant has higher lateral root density, early emerged lateral roots, and enhanced density of lateral root primordia	Li et al. (2015b)
			Increased O2 •‐ production in the mature area of the primary root containing lateral roots
AtRBOHE (A. thaliana)	Pollen development, involvement in late tapetal function	Unknown	Abnormal or reduced pollen in the mutant line	Xie et al. (2014)
			Increased expression in the tapetum
	Lateral root primordia formation	H2O2	The mutant shows delayed lateral root emergence	Orman‐Ligeza et al. (2016)
AtRBOHF (A. thaliana)	Primary root development, Casparian strip formation	H2O2	The mutant exhibits a shorter primary root with delayed Casparian strip formation	Morales et al. (2016)
	Lateral root development	H2O2	The mutant shows an increased number of lateral roots	Drerup et al. (2013)
AtRBOHH (A. thaliana)	Pollen development	H2O2	The mutant exhibits abnormal pollen tubes	Boisson‐Dernier et al. (2013)
				Lassig et al. (2014)
AtRBOHJ (A. thaliana)	Pollen development	H2O2	The mutant exhibits abnormal pollen tubes	Boisson‐Dernier et al. (2013)
				Lassig et al. (2014)
ZmRBOH7, ZmRBOH9, ZmRBOH14 (Zea mays)	Seed development	Unknown	Higher expression in the seed and endosperm	Zhang et al. (2023a)
ZmRBOH11 (Zea mays)	Stem development and growth	Unknown	Higher expression level in the stem and shoot apical meristem	Zhang et al. (2023a)

While RBOH abundances are regulated at the transcriptional, posttranscriptional and translational levels, their activation is mediated by PTMs, mainly phosphorylation, as well as Ca²⁺ binding. They may also be regulated by phosphatidic acid and protein‐protein interactions (Koo et al. 2017; Chapman et al. ²⁰¹⁹; George et al. ²⁰²³). RBOHs are also controlled by alternative splicing. For example, AtRBOHB is expressed in two splice variants differing in tissue‐ and development‐specific expression. The more prevalent variant is highly expressed in stress conditions (Lin et al. 2009; Van Ruyskensvelde et al. ²⁰¹⁸).

Auxin and ABA regulate RBOHs during development through hierarchies of intermediate messengers, including TFs, Ca²⁺, protein kinases, and phospholipids (Hu et al. 2020). The expression of RBOHs may be triggered by AUXIN RESPONSE FACTORS (ARFs), DNA‐binding proteins controlling the expression of many auxin‐related genes through binding to Auxin Response Elements (AuxREs) in promoter sequences. Indeed, four RBOH isoforms (ATRBOHC, ATRBOHD, ATRBOHI, ATRBOHJ) contain AuxREs. Similarly, seven Arabidopsis RBOH isoforms contain ABA‐responsive elements (ABREs), indicating their transcriptional regulation in response to ABA (Kaur and Pati 2016).

The interplay between auxin and RBOHs was most notably exemplified in root development, as described below. Little attention was also devoted to lateral bud outgrowth. The dynamics of lateral bud outgrowth number in tomato (Solanum lycopersicum) depend on the level of apoplastic ROS produced by SlRBOH1 and WHITEFLY INDUCED 1 (SlWFI1) and concomitant IAA biosynthesis, as shown by genetic suppression of these genes by virus‐induced gene silencing (Chen et al. 2016).

In primary roots, Arabidopsis single point knockdown mutants in RBOHC, rhd2‐1, rhd2‐2 and rhd2‐3, show reduced O₂ ^•‐ production in the QC and columella region, which is associated with reduced auxin levels. This contributes to phenotypic defects manifested by shorter and thicker primary roots and a reduced number of lateral roots compared to WT (Pasternak et al. 2023). In another study, auxin was reported to act upstream of RBOHs, as RBOHA, RBOHC, RBOHD and RBOHE are inducible upon synthetic auxin naphthaleneacetic acid treatment in Arabidopsis roots. RBOHs contribute to auxin‐controlled lateral root formation and development by ensuring the generation of O₂ ^•‐/H₂O₂ during this process. These ROS species are suggested to modulate cell wall physical properties, promoting lateral root formation (Orman‐Ligeza et al. ²⁰¹⁶); however, they may also exert signalling roles. Hence, RBOH‐mediated O₂ ^•‐/H₂O₂ accumulation triggered by auxin determined an expression of photomorphogenic gene LONG HYPOCOTYL 1 (HY1), which acts upstream of lateral root formation (Ma et al. 2014). Notably, H₂O₂ levels during lateral root formation and development are also regulated by specific peroxidase isoforms, such as PER7 and PER57 (Manzano et al. 2014).

RHO OF PLANTS (ROP) GTPases, proteins heavily implicated in auxin signalling, may activate RBOHs by protein‐protein interactions (Feiguelman et al. 2018). Unlike during osmotic stress response, ROP6 and RBOHD did not cluster in identical nanodomains, suggesting that ROP6 does not transduce the auxin signal toward ROS generation (Smokvarska et al. 2020; Kim et al. ²⁰²¹). However, during root hair formation, the RBOHC‐mediated O₂ ^•‐ production is promoted by FERONIA receptor‐like kinase (FER)‐ROP2 module. A root‐specific AtRBOHC is a canonical regulator of root hair formation, as ·OH, formed downstream of O₂ ^•‐ production, promotes root hair elongation (Foreman et al. 2003). Plasma membrane receptor kinase FER phosphorylates and activates ROP2 in response to auxin. ROP2 can activate RBOHC to produce O₂ ^•‐ in the apoplast, which is important for root hair formation (Duan et al. 2010). In addition, the co‐receptor LORELEI‐LIKE GPI ANCHORED PROTEIN 1 (LLG1) is required for the FER‐mediated O₂ ^•‐ production in response to auxin (Li et al. ^2015a; Tang and Guo ²⁰²⁵). To further demonstrate the complexity of RBOHC regulation, its activity is fine‐tuned during root hair growth through phosphorylation by CALCINEURIN B‐LIKE‐INTERACTING PROTEIN KINASE 26, which stimulates RBOHC activity in synergy with Ca²⁺ binding (Zhang et al. 2018).

In addition to these activation mechanisms, root hair growth is also preconditioned by sterol‐mediated AtRBOHC delivery to the apical plasma membrane (Kuběnová et al. 2022).

On the other hand, endocytosis and vesicular trafficking regulated by phosphatidylinositol (PtdIns) 3‐phosphate‐producing PtdIns 3‐kinase is inevitable for auxin‐induced O₂ ^•‐ production catalyzed by RBOHs and gravitropism (Joo et al. 2005). The crosstalk between phospholipid signaling and RBOHs is more comprehensively elucidated during plant immunity and ABA‐induced stomatal closure, as exemplified by the activation of RBOHD by phosphatidic acid (Zhang et al. 2009; Qi et al. ²⁰²⁴).

On the transcriptional level, the RBOHs regulation during root hair growth is ensured by ROOT HAIR DEFECTIVE SIX‐LIKE 4 (RSL4), an auxin‐responsive TF, controlled by several ARFs, including ARF5, ARF7, ARF8 and ARF19. RSL4 promotes the expression of RBOHC and RBOHH during root hair elongation (Mangano et al. 2017).

Similar to auxin, ABA can also induce the generation of O₂ ^•‐ through the activity of RBOHs. Specifically, ABA inhibits root elongation by regulating AtRBOHD and AtRBOHF in Arabidopsis. Mutants in AtRBOHD and AtRBOHF were characterised by longer cells in the elongation zone than WT in the presence of ABA (Kwak 2003; Jiao et al. ²⁰¹³). The ABA‐mediated regulation of the primary root elongation is closely associated to nitrate signalling and transporters in Medicago truncatula. LATERAL ROOT ORGAN DEFECTIVE/NUMEROUS INFECTIONS AND POLYPHENOLICS (LATD/NIP), also known as NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER FAMILY 1.7 (NPF1.7) is a nitrate transporter, which maintains ROS homeostasis by regulating MtRBOHA and MtRBOHC during root elongation. The mutant with reduced primary root elongation exhibits high expression of both RBOH isoforms, leading to O₂ ^•‐ accumulation, all manifestations being suppressed by exogenous ABA (Zhang et al. 2014).

The primary root growth is also regulated by MADS‐BOX TRANSCRIPTION FACTOR 25 (MADS25), whose expression is suppressed by ABA in Arabidopsis. MADS25 finely tunes O₂ ^•‐ levels in the elongation zone by modulating AtRBOHF and AtRBOHG expression, thereby impacting both primary root elongation and lateral root density (Xu et al. 2018). Another link between ABA and O₂ ^•‐/H₂O₂ was demonstrated in mutant lines in TF ABA‐INDUCED EXPRESSION 1 (AIN1). This TF is important in ABA signalling and maintaining O₂ ^•‐/H₂O₂. RNAi lines in AIN1 exhibited longer primary roots compared to WT during ABA treatment due to accelerated cell elongation and cell division. These defects were caused by increased O₂ ^•‐/H₂O₂ content due to higher expression of CAT1, CAT2, CAT3 and RBOHC, RBOHD and RBOHF and higher expression of WRKY40 and WRKY60, negative regulators in ABA signalling (Dong et al. 2021).

ABA is a master regulator of seed development and dormancy. It has been demonstrated that RBOHB is closely associated with seed development, and RBOHB mRNA exhibits two splicing variants (RBOHB‐α and RBOHB‐β) in embryos. Seed maturation is associated with increased expression of RBOHB‐α and O₂ ^•‐ production. Exogenous ABA blocks the transcription of RBOHB‐α mRNA, leading to a rapid drop in O₂ ^•‐ levels, and the seeds remain in dormancy (Müller et al. 2009).

Aside from the root, RBOH and RBOH‐produced O₂ ^•‐ play a role in reproduction. The Arabidopsis rbohe mutant shows reduced pollen number and pollen dysmorphia, indicating its role in pollen development (Xie et al. 2014). Further, pollen tube growth and fertilization involve the cooperation of RBOHH and J, as only the double rbohh/rbohj mutant shows altered pollen tube growth, likely due to impaired formation of O₂ ^•‐/H₂O₂ (Lassig et al. 2014).

Auxin and ABA, act in developmental regulatory networks in synergistic manner. This crosstalk is often mediated through auxin and ABA‐responsive TF interactions (Ortiz‐García et al. ²⁰²³). Auxin signalling promotes ABA signalling, leading to enhanced seed dormancy and inhibition of seed germination through stimulation ABA signalling by inducing ARF10 and ARF16‐mediated ABI3 activation (Liu et al. 2013). ABI3 has also been described to be negatively regulated by the auxin signalling repressor IAA8, whose accumulation promotes seed germination (Hussain et al. 2020). The interplay can be regulated through complex loops. For instance, the ABI3‐ERF1 module can mediate crosstalk to regulate lateral root emergence, with ABI3 activating ERF1 and ERF1, in turn, activating ABI3. Physical interactions of ABI3 and ERF1 reduce cis‐element binding activities of both and thus attenuating the expression of PIN1, AUX1, ARF7 and ABI5 (Zhang et al. ^2023b). ABA and auxin interaction is linked also to the growth and trajectory of primary root. Here, ABA suppresses shootward auxin transport by reduction of the membrane abundance of PIN2 leading to reduced growth rate of primary roots, disruption of gravitropism and proper distribution of auxin (Xie et al. 2021).

Expectedly, ABA and auxins participate on ROS homeostasis during development. ABA affects auxin distribution and PLETHORA protein stability during root growth through the production of ROS in root tips mitochondria (Yang et al. 2014). Notably, alterations in genes encoding antioxidant enzymes may also have consequences in auxin and ABA homeostasis. Knockout of the APX6 gene resulted in an increase in ROS levels, which contributed to an elevation in both ABA and auxin levels, which was associated with an increase in ABI4 expression. These changes resulted in the inhibition of seed germination (Chen et al. 2014). Taken together, fulfillment of developmental programs is determined by controlled tissue and organ‐specific spatiotemporal RBOHs expression, subcellular distribution and relocation, and posttranscriptional, translational, and posttranslational regulation.

4. Regulation of SODs and Their Roles in Developmental Processes

The О₂ ^•– scavenging by SODs is accompanied by the production of H₂О₂, which is further decomposed by peroxidases, catalases, the ascorbate‐glutathione cycle, peroxiredoxins, glutaredoxins, and thioredoxins (Chapman et al. 2019; Dvořák et al. ^2021b). О₂ ^•– might be scavenged also nonenzymatically by ascorbate (Njus et al. 2020), tocopherol (Kumar et al. 2021), or by its reaction with H₂О₂ (in the Haber‐Weiss reaction), nitric oxide (NO) (Speckmann et al. 2016), or H₂S (Filipovic and Jovanović 2017). Nevertheless, the most potent scavengers are SODs and ascorbate, which help maintain this molecule at optimal concentrations (Karpinska and Foyer 2024). Little is known so far about the complementation of various О₂ ^•– ‐decomposing routes during developmental processes.

SODs are specific enzymatic antioxidant players because they maintain cellular equilibrium between О₂ ^•– and H₂О₂. Their catalytic activity must be precisely regulated to maintain О₂ ^•– and H₂O₂ concentrations at physiological levels and ensure their signalling functions (Karpinska and Foyer 2024). The activity of SOD requires a metal ion at its active site; in plants, this includes iron for FeSOD, copper and zinc for Cu/ZnSOD, and manganese for MnSOD. Each of these isozymes is encoded by several isoforms with specific localisation and expression features (Pilon et al. 2011). In Arabidopsis, three FeSODs (FSD1, FSD2, and FSD3), two MnSODs (MSD1 and MSD2), and three Cu/ZnSODs (CSD1, CSD2, and CSD3) isoenzymes have been experimentally confirmed (Kliebenstein et al. 1998; Chen et al. ²⁰²²). CSDs evolved independently of FSDs and MSDs due to increased oxygen levels during or after the Great Oxygenation Event (Dreyer and Schippers 2019). Their distribution across plant species reflects evolutionary divergence as well as specific physiological and metabolic requirements and sources of ROS production in basal metabolism (Dreyer and Schippers 2019). The subcellular distribution of Arabidopsis SOD isoforms correlates with the place of О₂ ^•– production: MSD1 is responsible for the scavenging of mitochondrial О₂ ^•– (Kliebenstein et al. 1998); CSD1 and FSD1 are both localised in the cytosol, and FSD1 is also present in the nucleus (Kliebenstein et al. 1998; Dvořák et al. ^2021a). In chloroplasts, the main SODs are FSD1 in the stroma and CSD2, FSD2, and FSD3 in thylakoids (Kliebenstein et al. 1998; Myouga et al. ²⁰⁰⁸; Dvořák et al. ^2021a). CSD3 is so far the sole SOD isoform detected in peroxisomes (Huang et al. 2012). Recently, MSD2 was identified as the first SOD in Arabidopsis localised to the apoplast (Chen et al. 2022).

Expression of SODs is developmentally controlled. In Arabidopsis, FSD1 has the highest expression among all isoforms, peaking during seed germination and early rosette development. On the other hand, CSD1 and CSD2 expression are lower at early developmental stages and increase after flowering (Pilon et al. 2011). Peroxisomal CSD3 is expressed at substantially lower levels, with less variation, most prominent in flowers and seeds (Chu et al. 2005; Zhou et al. ²⁰²²). FSD2 and FSD3 expression were shown to peak in the later stages of rosette development and flower development, when they are likely essential for protecting developing chloroplasts (Myouga et al. 2008). MSD1 shows moderate expression throughout all developmental stages (Pilon et al. 2011). In situ hybridisation revealed that most of the 7 SOD isoforms (except FSD3) are preferentially expressed in the differentiating peripheral zone of the shoot apical meristem (SAM) (Zeng et al. 2017). This indicates that specific developmental processes are more demanding for SOD activity. Nevertheless, as published for yeasts or mammalian cells, SOD may also exert functions beyond О₂ ^•– decomposing activity (Tsang et al. 2014).

SOD mutants show varying developmental phenotypes. FSD2 and FSD3 are uniquely chloroplastic, and form heteromeric complexes, presumably to protect chloroplast DNA. Single fsd2 and fsd3 mutants exhibit stunted habitus and pale green leaves (Myouga et al. 2008). Both isoforms are components of the plastid‐encoded RNA polymerase complex, suggesting a possible regulatory role in chloroplast gene expression (Pfannschmidt et al. 2015; Lee, Joung, Kim, Do Choi & Jang ²⁰¹⁹), a role that remains to be verified. This function, together with О₂ ^•– decomposition, may contribute to their pronounced and unique phenotype, which is not observed in other SOD mutant lines (Myouga et al. 2008).

Despite the obvious developmental expression dynamics, Arabidopsis fsd1 mutants do not exhibit pronounced developmental phenotypes, suggesting that FSD1 is conditionally essential. Knockout fsd1 mutants have shown mild defects in lateral root formation. In contrast, GFP‐tagged FSD1, expressed under its native promoter, showed increased signal in emerging lateral root primordia and growing root hairs. Also, it exhibited a tissue‐specific expression pattern in the root tip and showed preferential accumulation in endodermis/cortex initials and central columella cells in root tips (Dvořák et al. ^2021a). Hence, FSD1 may contribute to the regulation of redox status in dividing cells, such as root initials, as suggested by the FSD1‐GFP signal distribution (Figure 2). Redox homeostasis, which acts downstream of the auxin transport, is also known to maintain root meristematic activity a QC organisation (Foyer 2018). Intriguingly, the FSD1 tissue‐dependent expression pattern (Figure 2) largely correlates with auxin maxima in the root tip (Petersson et al. 2009) and with О₂ ^•– maxima (Dunand et al. 2007). Furthermore, FSD1 might also contribute to the regulation of two GRAS‐type TFs, SCARECROW and SHORTROOT, during endodermis formation. The high expression of FSD1 was detected in protoplasts expressing endoplasmic reticulum‐targeted GFP under the control of the SCR promoter, and this expression was elevated in salt‐stressed protoplasts (Geng et al. 2013). Considering this, FSD1 may be involved in maintaining redox homeostasis in the endodermis/cortex initials of the root tip. Next, prominent expression maxima were observed at the site of endosperm rupture using in vivo light‐sheet microscopy; however, the mutant shows a conditional germination delay phenotype only under salt stress (Dvořák et al. ^2021a). It remains to be addressed which subcellular FSD1 pool is essential for the conditional developmental functions of this intriguing protein.

Figure 2.

Overview of FSD1‐GFP tissue‐specific localisation in primary root apex with root cap, quiescence center and part of the meristem. (A‐C) Seedlings expressing FSD1‐GFP were stained with FM4‐64 and observed under a confocal laser scanning microscope. Channels: green ‐ FSD1‐GFP (A); magenta ‐ FM4‐64 (B); overlay (C). Scale bar: 20 μm. (Dvořák et al. ^2021a). [Color figure can be viewed at wileyonlinelibrary.com]

Unfortunately, detailed data on the tissue‐specific expression of other SOD isoforms have not been published to date. Considering the root phenotypes, mitochondrial MSD1 may be the main regulatory SOD isoform, as an antisense Arabidopsis line with suppressed MSD1 exhibited shorter roots, and had lower fresh weight, presumably due to a specific disturbance of mitochondrial redox homeostasis (Morgan et al. 2008). MSD1 is also highly expressed during the development of female reproductive organs, which correlates with the mitochondrial О₂ ^•– pattern. By controlling the accumulation of mitochondrial ROS in the central cell nucleus and the micropylar region of the embryo sac at stage FG6‐FG7, MSD1 regulates PCD of antipodal cells (Martin et al. 2013). Another MnSOD form, apoplastic MSD2, also participates in root development, as MSD2 mutants displayed defects in root growth in the dark, with a more extended elongation zone (Chen et al. 2022). C‐terminally fused MSD2 with the fluorescent mCherry tag expressed under the UBQ10 promoter, showed that it is highly abundant at the hypocotyl‐root junction and root tip and root differentiation zone, where it presumably regulates the distribution of О₂ ^•– and H₂О₂ (Chen et al. 2022). The emerging question is whether MSD2 participates in the dismutation of apoplastic O₂ ^•‐ produced by RBOHs, and whether its absence disrupts ROS signalling in both developmental processes and the activation of defense mechanisms.

Although Arabidopsis CSDs have primarily been associated with stress responses, their potential role in developmental regulation remains unexplored. Tobacco Cu/ZnSOD1 has been implicated in pollen‐tube growth, as antisense knockdown of NtCu/ZnSOD1 resulted in pollen tubes with reduced length (Hafidh et al. 2012). In tomato, chloroplastic Cu/ZnSOD isoforms show higher expression in the apical meristem and young leaves compared with the root (Perl‐Treves and Galun ¹⁹⁹¹). In pine and aspen, extracellular Cu/ZnSODs have been identified in the secondary cell wall, where their functions remain unclear (Schinkel et al. 2001; Karpinska et al. ²⁰⁰¹). Extracellular Cu/ZnSOD is involved in primary and secondary cell wall formation in cotton, by the regulation of ROS levels (Kim et al. 2008). This clearly indicates that the developmental roles of Cu/ZnSOD isoforms should be examined more thoroughly in future research, particularly considering that they represent the newest evolutionary variants of plant SODs.

Given the fluctuations in expression of different SOD isoforms during growth and development, their regulation is essential and can occur at multiple levels. Primarily, the expression of CSDs and FSD1 critically depends on the bioavailability of Cu and Fe. The TF SQUAMOSA PROMOTER BINDING‐LIKE PROTEIN 7 (SPL7), a member of the prominent SQUAMOSA promoter binding protein (SBP) family, is the central regulator of Cu deficiency responses in Arabidopsis, binding to GTAC motifs in the promoters of its target genes (Yamasaki et al. 2009; Shikata et al. ²⁰⁰⁹). SPL7 directly induces FSD1 expression in conditions of Cu deficiency, as well as miRNA398 to downregulate CSD1, CSD2, and COPPER CHAPERONE FOR SUPEROXIDE DISMUTASE (CCS) (Yamasaki et al. 2007; Schulten et al. ²⁰²²). Hence, this compensatory mechanism ensures the substitution of isoforms under certain conditions, which is particularly vital during the stress response (Melicher et al. 2022). In general, miR398 and Cu‐dependent regulation of Cu/ZnSODs are conserved in the majority of plant species, such as Medicago truncatula (Lelandais‐Brière et al. ²⁰⁰⁹), Arachis hypogea (Zhao et al. 2010), and Glycine max (Song et al. 2011).

In addition to SPL7, plant SODs may be subject to transcriptional control by some other TFs; however, experimental evidence of TF binding to SOD promoter sequences has not been reported to date. Nevertheless, upstream of transcriptional regulation lies the necessity for its induction, which, during development, is orchestrated by hormonal signalling. Plant hormones such as auxin and ABA could affect SODs expression, for example, CSD1 and CSD2 mRNA levels were increased during 4 h of ABA treatment, but dropped after 24 h (Jia et al. 2009). As a consequence, changes in SOD expression could contribute to ROS homeostasis during hormone‐mediated developmental processes. For example, during germination, O₂ ^•‐ and H₂O₂ homeostasis is essential for breaking seed dormancy maintained by high ABA levels in the seed coat (Bailly et al. 2008). Peroxisome‐derived ROS (mainly O₂ ^•‐ and H₂O₂) also contribute to seed germination and postgerminative growth (Bykova and Igamberdiev 2025), coordinating lipid mobilisation and establishing metabolic autonomy in the developing seedling (Pan et al. 2019). One of the proteins essential for seed germination is ABA AND ROS SENSITIVE 1 (ARS1), which may regulate redox balance during the breaking of seed dormancy, regarding peroxisome‐derived ROS. In the ars1 mutant, elevated redox levels result from reduced CDS3 expression, suggesting its role in peroxisomal O₂ ^•‐ and H₂O₂ homeostasis during seed germination (Baek et al. 2015). However, the precise mechanisms by which ARS1 regulates CDS3 expression remain unclear. Moreover, in rice, overexpression lines of peroxisomal OsCu/ZnSOD2 and cytosolic OsCu/ZnSOD3 showed higher seed dormancy and elevated ABA content, suggesting a conserved role for Cu/ZnSOD and peroxisome‐derived O₂ ^•‐ and H₂O₂ in ABA‐mediated regulation of seed longevity (Zheng et al. 2024).

Another connection of ABA‐induced H₂O₂ and SOD was shown in maize leaves (Hu et al. 2005). Histochemical and cytochemical methods revealed ABA‐induced apoplastic H₂O₂ accumulation in mesophyll. Isolated chloroplast and cytosol fractions showed a significant increase in SOD activity after ABA treatment. The authors suggest that apoplastic H₂O₂ could coordinate the overall activity of chloroplastic and cytosolic SOD, possibly by triggering redox signalling and up‐regulating defence system components after being transported from the apoplast to the cytosol via aquaporins (Mittler 2002; Hu et al. ²⁰⁰⁵). However, the mechanisms through which imported H₂O₂ exerts its functions across different organelles and subcellular compartments remain to be elucidated. Further, the apoplastic O₂ ^•‐ and H₂O₂ metabolism, regulated by secretory MSD2, affects the timing of flower abscission by regulating NO and ABA signalling (Lee et al. 2022). Genetic studies showed that MSD2 acts upstream of the receptor‐like kinase HAESA (HAE) and its cognate peptide ligand INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) involved in floral organ abscission. Thus, MSD2 connects ROS management with ABA‐mediated developmental signalling (Lee et al. 2022). SODs also respond to fluctuations in levels of auxin which could either induce SOD expression (Crowell and Amasino 1991), or alter enzyme activity (Takáč et al. 2016). In tomato, exogenous auxin application caused a decrease in O₂ ^•‐/H₂O₂ levels in the meristem and root cap, due to elevated activities of Cu/ZnSOD and CAT, which resulted in root growth inhibition (Tyburski et al. 2009). This result correlates with another study, which found that Arabidopsis knockdown‐SOD (KD‐SOD) plants showed higher H₂O₂ levels in the root apical meristem (RAM). In contrast, auxin levels were comparable in the QC region and the columella, but were higher in the inner tissue in the differentiation zone of roots. Moreover, KD‐SOD plants showed shorter primary roots and lower abundance of PIN1, PIN2, and PIN4 proteins, suggesting that elevated H₂O₂ levels disrupt auxin distribution, thereby reducing cell proliferation and delaying primary root growth (Pasternak et al. 2023). The auxin‐O₂ ^•‐ crosstalk is linked also to HEAT SCHOCK PROTEIN 70‐1 (HSC70‐1) in M. truncatula. Dysfunction of MtHSC70‐1 leads to decreased expression of genes involved in auxin biosynthesis and polar transport, which causes defective embryo development and severe growth inhibition. However, the introduction of mitochondrial MSD1 into mthsc70‐1a plants caused a decrease in O₂ ^•‐ and H₂O₂ in the root, reverting a wild‐type root phenotype (Shen et al. 2022). Altogether, it points to the connection between mitochondrial ROS homeostasis mediated by MSD1 and polar auxin transport via PIN in the cytosol, but still, the mechanism of this interconnection and the ROS mitochondrial retrograde regulation is yet to be investigated.

5. Mechanisms Linking O₂•‐ Production With Scavenging

Proper developmental programs depend on precise spatial and temporal control of ROS production and scavenging. Spatially, tissues and cells must maintain specific ROS distributions while allowing for rapid relocation when needed. Appropriate ROS levels must also be kept in the correct subcellular compartments. To sustain polar growth, ROS must accumulate in specific cell regions, reflecting their polar distribution. Achieving this requires tight coordination between ROS production and scavenging.

So far, only a few studies have reported on the enzymes simultaneously contributing to ROS production and scavenging during developmental processes. Stem cell maintenance in the Arabidopsis SAM is controlled by the spatial distribution of O₂ ^•‐ and H₂O₂. O₂ ^•‐ preferentially accumulates in stem cells, largely through RBOHD and RBOHF activity, with mitochondrial ROS also contributing, and regulates WUSCHEL (WUS) expression (Considine and Foyer 2024). To sustain high O₂ ^•‐ levels, most SOD isoforms are repressed in expression, while O₂ ^•‐ may promote RBOH expression (Zeng et al. 2017; Considine and Foyer ²⁰²⁴). The isoform‐nonspecific SOD repression raises questions about how chloroplastic (FSDs or CSD2) or mitochondrial (MSD1) O₂ ^•‐ influences pivotal stem‐cell determinants. In contrast, H₂O₂ accumulates in the differentiating peripheral zone and may, by yet unknown mechanism, inhibit RBOH activity to gate O₂ ^•‐ production. Recent work (Wang et al. 2025) reveals that DNA demethylase REPRESSOR OF SILENCING 1 (ROS1), a Fe‐S cluster‐containing protein, is a direct target of O₂ ^•‐ and the oxidation of the Fe–S clusters leads to its activation, linking O₂ ^•‐ signalling to epigenetic regulation. Thus, high accumulation of O₂ ^•‐, caused by suppression of SODs in stem cells, promotes ROS1‐mediated DNA demethylation, integrating redox and epigenetic control of WUS expression. An emerging question is the source of O₂ ^•‐ in the nucleus that triggers such an epigenetic modification. A recent publication predicted that some RBOHs may be partially localised in the nucleus and could serve as a source of nuclear O₂ ^•‐ (Karpinska and Foyer 2024), justifying also the presence of FSD1 nucleus (Dvořák et al. ^2021a). Another possible explanation is that ROS1 is activated in the cytosol by O₂ ^•‐ and then relocalises to the nucleus, a question that still remains to be answered. In mammalian systems, H₂O₂ was reported to possess a rather self‐propagating ability by activation of RBOH to enhance O₂ ^•‐ production (Cai 2005; Mu et al. ²⁰¹⁰; Lin and Wang ²⁰¹²). This, therefore, represents another mechanism to modulate O₂ ^•‐/H₂O₂ distribution.

In the Arabidopsis root tip, H₂O₂ promotes periclinal cell division in the endodermis/cortex initials. The levels of H₂O₂ are promoted by SHORTROOT, by activating RBOHE and RBOHF. Simultaneously, the salicylic acid signalling pathway is enhanced to repress CAT2 expression (Li et al. 2020). The high H₂O₂ levels might be maintained by FSD1, since it shows maxima in endodermis/cortex initials (Dvořák et al. ^2021a).

Little attention is currently dedicated to delineating the signals and regulatory mechanisms that integrate and synchronize the ROS‐producing and decomposing mechanisms during plant developmental processes. In the next sections, we discuss possible activation of SODs and RBOHs by oxidative posttranslational modifications, transcriptional co‐activation of RBOHs and SODs as well as co‐regulation of RBOHs and SODs by protein‐protein interactions.

5.1. Regulation of SOD and RBOHs by Oxidative PTMs

SODs could be potentially activated or inactivated by oxidative PTMs mediated by H₂O₂/•OH generated as indirect products of RBOH activity. Since the RBOHs generate O₂•‐/H_{2 O2}, which modulate the cellular redox environment, they can influence the reactivity of protein cysteine residues, thereby indirectly affecting S‐nitrosylation mediated by NO. Through this ROS–NO crosstalk, RBOH activity can regulate S‐nitrosylation of key signaling proteins, including possibly SODs, fine‐tuning processes such as stress responses, defense signaling, and developmental processes (Parveen et al. 2022; Borrowman et al. ²⁰²³; Bykova and Igamberdiev ²⁰²⁵). We took advantage of pCysMod prediction tool (Li et al. 2021) and screened for the presence of redox‐active cysteines in the amino acid sequences of Arabidopsis (a representative dicot) and Oryza sativa (a representative monocot) SOD isoforms (Supporting Table 1, 2). Although we have found numerous redox‐active cysteines in almost all AtSOD isoforms, iCysMod database (Wang et al. 2021) and Plant PTM Viewer (Willems et al. 2019) contain only scarce records of experimentally found SOD oxidation (Supporting Tables 1, 2). H₂O₂‐mediated cysteine oxidation was identified for chloroplastic CSD2 on C140 (Slade et al. 2015; Supporting Tables 1, 2) and CSD2 may also undergo S‐nitrosylation at C119 (Hu et al. 2015), which is consistent with prediction, indicating a high likelihood of S‐nitrosylation at this residue. Other redox‐sensitive cysteines have been identified at C208 in CSD2 as well as C56 and C145 in CSD1 (Doron et al. 2021). C145 is predicted to be S‐nitrosylated with a high likelihood (Supporting Table 1). Since SODs are highly conserved across all species (Dreyer and Schippers 2019), these Cys residues. This role was assigned for C57 and C146 in human SOD1, where they help maintain structural integrity and prevent protein aggregation (Álvarez‐Zaldiernas et al. ²⁰¹⁶). The oxidation of AtCSD3 on C62 is ABA‐dependent and is enhanced in Gβ‐protein null mutant agb1 (Smythers et al. 2022). Using redox proteomic analysis, it was found that CSD1 and CSD2 undergo a more pronounced reversible oxidation in Arabidopsis in response to high light levels. Moreover, the oxidation rate increased over time (Hou et al. 2024).

The results obtained from the Plant PTM Viewer are particularly intriguing, as they suggest that N‐terminal proteolysis, N‐terminal acetylation (NTA), and lysine acetylation may be frequent post‐translational modifications in AtSODs, as identified across multiple proteomic studies (Supporting Table 2). Interestingly, the NTA of Arabidopsis FSD1 and CSD1, was detected in proteomic studies (Bienvenut et al. 2012; Soh et al. ²⁰²⁰). CSD2, CSD3, and MSD1 are also candidates for this modification, as they contain amino acids compatible with NTA at their N‐α‐amino groups (Figures 3, 4). NTA is an irreversible co‐ and post‐translational modification involving the enzymatic transfer of an acetyl group from acetyl‐CoA to protein N‐α‐amino groups. This modification, catalyzed by N‐terminal acetyltransferases, is ubiquitous across all domains of life, with an increasing proportion of proteins modified in more complex organisms (Øye et al. 2025). NTA can regulate protein stability, folding, complex assembly, subcellular localisation, and protein‐protein interactions in plants (McTiernan et al. ²⁰²⁵). In Arabidopsis, proteomic analyses revealed that loss of NTAs leads to protein destabilisation through a novel nonAc/N‐degron, highlighting the widespread impact of this modification on proteome homeostasis (Linster et al. 2022). This raises intriguing questions about the regulatory role of NTA in AtSODs function and warrants further investigation, particularly for FSD1 and CSD1, where this modification has been experimentally observed.

Figure 3.

Sequence alignment of Arabidopsis and rice Cu/Zn superoxide dismutases (SODs). Multiple sequence alignment of AtCSD1 (AT1G08830), AtCSD2 (AT2G28190), AtCSD3 (AT5G18100) and OsCSD1 (LOC_Os03g22810); OsCSD2 (LOC_Os08g44770); OsCSD3 (LOC_Os03g11960); OsCSD4 (LOC_Os07g46990). Conserved residues across all sequences are marked with asterisks (*). A colon (:) denotes positions with strongly similar properties, and a period (.) indicates weakly similar properties. Cysteines that could potentially undergo redox modifications are highlighted in bold red, and amino acids (Ala, Val) that could undergo N‐terminal acetylation are highlighted in pink. Amino acids responsible for maintaining the structure and function of the active site are highlighted in blue; involved in metal‐binding in green, and involved in superoxide binding in brown. Alignments were performed using Clustal Omega. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4.

Sequence alignment of Arabidopsis and rice Fe and Mn superoxide dismutases (SODs). Multiple sequence alignment of AtFSD1 (AT4G25100), AtFSD2 (AT5G51100), AtFSD3 (AT5G51100), AtMSD1 (AT3G10920), AtMSD2 (AT3G56350) and OsFSD1 (LOC_Os06g05110); OsFSD2 (LOC_Os06g02500); OsMSD (LOC_Os05g2585). Conserved residues across all sequences are marked with asterisks (*). A colon (:) denotes positions with strongly similar properties, and a period (.) indicates weakly similar properties. Cysteines that could potentially undergo redox modifications are highlighted in bold red, and amino acids (Ala, Val) that could undergo N‐terminal acetylation are highlighted in pink. Amino acids responsible for maintaining the structure and function of the active site are highlighted in blue; involved in metal‐binding in green, and involved in superoxide binding in brown. Alignments were performed using Clustal Omega. [Color figure can be viewed at wileyonlinelibrary.com]

There is very limited experimental evidence on oxidative PTMs of OsSODs. A recent S‐nitrosoproteomics study on two rice cultivars, Dongjin (DJ, resistant) and Nipponbare (NIP, susceptible), identified induced S‐ nitrosylation in VACGIIGLQG peptide of OsSOD, while this PTM significantly affected total SOD activity (Munir et al. 2025). Interestingly, this S‐ nitrosylation was also predicted by pCysMod with high confidence (Supporting table 1) and occurs on a conserved Cys145 residue in both OsCSD1 and OsCSD4 (Figure 3). This residue is responsible for protein stabilisation, as described previously for AtCSD1 and human SOD1, thus highlighting the evolutionarily conserved function of this SOD1 Cys, whose role in plants appears promising for future studies. Of note, several OsSODs were predicted to have other Cys that could be potential targets of S‐nitrosylation (Supporting Table 1). On the contrary, results from Plant PTM Viewer revealed diverse PTMs of OsSODs, including 2‐hydroxyisobutyrylation, succinylation, crotonylation, malonylation, and acetylation, many of which are not present in AtSODs (Supporting Table 2). On the other hand, NTA was absent, even though sequence alignment revealed several conserved alanine residues at the N‐α‐amino position of OsSODs (Figure 3). This may be caused by limited dataset coverage in crop species.

In summary, an alignment of AtCSD and OsCSD isoforms revealed 2 conserved cysteine residues whose oxidation have been experimentally proved (Figure 3). These have not been identified in AtMSD and AtFSD isoforms (Figure 4), that evolved before the Great Oxidation Event, at a time when the Earth′s atmosphere was deficient in substantial molecular oxygen. This likely determined the lower capability of redox modification‐mediated regulation of these isoforms. Although the influence of oxidative PTMs on recombinant AtSOD prepared in the bacterial expression system was examined in vitro (Holzmeister et al. 2015), the functional relevance of such PTMs has not yet been elucidated in plants. In mammalian cells, oxidative PTMs of SODs are more common, and modifications such as oxidation to sulfonic and sulfenic acids, as well as S‐glutathionylation, have been associated with protein misfolding or conformational changes (Banks and Andersen 2019).

The roles and impacts of various PTMs of RBOHs across a wide range of plant species have been comprehensively reviewed in recent studies (Castro et al. 2021; Zhang et al. ²⁰²⁵) and are therefore not described in detail here. Overall, two oxidative PTMs exerting direct effects on RBOH activity have been experimentally characterized in plants: S‐nitrosylation of AtRBOHD at Cys890, which negatively regulates its activity (Yun et al. 2011), and persulfidation at Cys825 and Cys890, which enhances O₂ ^•‐ production by AtRBOHD (Shen et al. 2020). pCysMod‐based prediction revealed several additional potential sites for oxidative PTMs in both AtRBOHs and OsRBOHs, primarily S‐nitrosylation (Supporting Table 1). Some of these sites exhibited a high likelihood of modification and can be considered as promising targets for further investigation, as this type of modification has previously been shown to directly affect RBOH activity. On the other hand, analysis using the Plant PTM Viewer revealed predominantly phosphorylation events in both AtRBOHs and OsRBOHs, some of which have been experimentally validated (Supporting Table 2). Interestingly, NTA was also identified as a potential PTM in AtRBOHA and AtRBOHJ, which, as suggested above for AtSODs, may be of interest for further studies and could play a role in the co‐regulation of these enzymes.

These findings suggest that O₂ ^•‐ scavenging by SODs may be interconnected with its production by RBOH through redox‐based PTMs. Considering the limited capability of O₂ ^•‐ to oxidize protein substrates, SODs are potentially oxidized by H₂O₂ derived from O₂ ^•‐ decomposition. The O₂ ^•‐ scavenging would serve as a feedback activation mechanism in such a model.

However, this interplay depends on the shared apoplastic localization of both enzymes and the presence of redox‐active amino acid residues. Consequently, this mechanism is likely isoform‐specific.

5.2. Co‐Activation of SODs and RBOHs Expression by Shared Transcription Factors

Phytohormones and peptide ligands are pivotal regulators of plant developmental processes, which may regulate the expression of RBOHs as well SODs, by activation of TFs (Mangano et al. 2017; Yamada et al. ²⁰²⁰; Hastwell et al. ²⁰²⁴). TFs can potentially induce the expression of SOD and RBOH simultaneously (Figure 5A). Therefore, we screened the promoter sequences of these genes for shared cis‐regulatory elements. Applying two bioinformatic databases, PlantCARE and Agris, which contain information on plant cis‐acting regulatory elements, enhancers and repressors, we demonstrate potential cis‐elements common to almost all isoforms of RBOH and SOD in Arabidopsis (Figure 5B, Supporting Table 3).

Figure 5.

Transcriptional co‐regulation of NADPH oxidase‐mediated superoxide anion production with its scavenging by superoxide dismutases, governed by auxin and abscisic acid signaling. (A) Schematic model depicting how ABA and auxin influence the expression of NADPH oxidases and SODs. In the presence of auxin, the hormone binds to the TIR1 receptor, increasing TIR1's affinity for Aux/IAA repressors. Subsequently, Aux/IAA proteins are tagged with ubiquitin for degradation via the 26S proteasome. This results in the liberation of ARFs, thus allowing ARFs to promote transcription of RBOHs and SODs. On the other hand, in the presence of ABA, the ABA receptor PYR/PYL/RCAR interacts with PP2C, blocking phosphatase action and releasing SnRK2 from negative regulation. The activated SnRK2 phosphorylates ABA‐responsive transcription factors, which in turn induce the transcription of RBOHs and SODs. (B) Heat map showing the abundance of cis elements in the promoter sequences of individual SOD and RBOH isoforms. The presence of cis elements was evaluated by PlantCare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) between −2000 and −1 bp upstream of the start codon of each gene sequence. The number of cis elements the promoter sequences in indicated by color according to the color key in the right. [Color figure can be viewed at wileyonlinelibrary.com]

TATA‐box and CAAT‐box motifs are the most common cis‐elements found in bioinformatic analysis. They are common to both RBOHs and SODs. TATA‐box (TATAAA) is recognised by TATA‐binding protein (TBP), a component of the Transcription factor II D (TFIID) complex. Similarly, CAAT‐box (CCAAT) acts as a binding site for TFs, such as CCAAT‐enhancer‐binding proteins (C/EBPs). Both of these cis‐elements are very important for the initiation of transcription, affecting the efficiency and rate of transcription (Singh et al. 2014; Savinkova et al. ²⁰²³).

Another motif occurring in the promoter sequence of all selected enzymes is the conserved ABRE (ACGTGG/TC). They are recognized by TFs activated during stress conditions, such as dehydration, high salinity, or low temperatures. ABRE elements are also the main calcium‐regulated promoter motifs. They control the expression of genes responding to ABA‐induced signaling pathways, regulated by calcium signals (Whalley et al. 2011; Huda et al. ²⁰¹³).

ROS production and release depend on a mechanism that may consist of the activities of individual cis‐elements located upstream of the region of the contributing genes. These motifs were previously divided into two groups: common ROS‐related and ROS‐specific cis‐elements (Gadjev et al. 2006; Petrov et al. ²⁰¹²). The ROS‐specific motifs in RBOHs and SODs are represented by G‐box (CACGTG) and W‐box (TTGACC) elements. The former is frequent in genes involved in hormone signalling and light response, whereas the W‐box is recognised by TFs called WRKY, that play an essential role in defense against biotic and abiotic stress (Rushton et al. 2010).

There is very little experimental evidence for specific TFs binding to the G‐box or W‐box that are common to RBOHs and SODs. Kang et al. (2020) found that transcript levels of CSD1, CSD2, FSD3, and RBOHD were significantly increased in Arabidopsis plants overexpressing the Hevea brasiliensis HbWRKY82. Another TF from the WRKY family, specifically AtWRKY57, overexpressed in Oryza sativa increased the expression levels of OsCSD1 and OsCSD2 (Jiang et al. 2016). Transgenic Arabidopsis lines overexpressing Kandelia obovate KoWRKY40 exhibit increased transcript levels of MSD1 (Fei et al. 2022).

NTL4, a G‐box‐binding NAC TF can regulate ROS production by binding it to the AtRBOHC and AtRBOHE promoter sequences (Lee et al. 2012). In tobacco plants, the bHLH123 controls defense mechanisms against abiotic stress conditions through binding to the G‐box element of the RBOHE gene (Liu et al. 2021).

Finally, MYB and MYC elements, occurring in both RBOHs and SODs promoters, are motifs recognised by MYB and MYC TFs. These transcription factors are key regulators of flavonoid biosynthesis (Nakabayashi et al. 2014) and cold tolerance (Ohta et al. 2018).

These results suggest that RBOHs and SODs share many cis‐elements that are recognised by a wide range of TFs, involved in many biological processes, such as development, responses to phytohormones and environmental stress conditions. Nevertheless, other mechanisms, including the transcriptional mediator complex, may also be efficient in the expression control of ROS homeostasis. PHYTOCHROME AND FLOWERING TIME1 (PFT1), also known as MED25, a subunit of this complex, regulates ROS distribution in root and root hairs by activating a subset of H₂O₂‐producing class III peroxidases (Sundaravelpandian et al. 2013). Nevertheless, the question of whether it might target RBOHs and SODs simultaneously remains to be revealed.

5.3. Protein‐Protein Interactions

ROS production and scavenging might be coordinated spatially and temporally by proteins that bind to SOD and RBOH simultaneously. Indeed, antioxidant enzymes, particularly SODs, interact with a wide range of proteins, which mediate their activation, stabilisation or folding. The protein pairs jointly participate in diverse functions, including ion homeostasis or retrograde signalling (Melicher et al. 2022). An interacting partner of Arabidopsis SODs, more specifically FSD1 and CSD1, is RECEPTOR FOR ACTIVATED PROTEIN KINASE C (RACK1) (Guo et al. 2019; Melicher et al. ²⁰²⁵), demonstrated the ability to bind also RBOHs. In rice, OsRACK1B interacts with the N‐terminus of RBOHD and OsRACK1B overexpression results in increased H₂O₂ accumulation in pollen, negatively affecting its development (Rahman et al. 2022). The ROS‐stimulating effect was also observed for OsRACK1A, which interacts with RBOHB during immune responses (Nakashima et al. 2008). Notably, RACK1A is phosphorylated and undergoes dimerization upon oxidative stress (Sabila et al. 2016), which might trigger protein‐protein interactions. RACK1 also interacts with subunits of heterotrimeric G proteins (Olejnik et al. 2011). AlphaFold2‐Multimer modeling of the AtRBOHC–AtRACK1A–AtFSD1 complex, reveals an amino acid‐abundant protein–protein interaction interface between AtRACK1A and AtRBOHC (Figure 6A), whereas fewer amino acids interact in the interface of AtRACK1A and AtFSD1 (Figure 6A, B). AtRBOHC (Kuběnová et al. 2022), AtFSD1 (Dvořák et al. ^2021a), and AtRACK1A (Melicher et al. 2022) accumulate at the growing root hair tip, suggesting possible roles for this module in controlling ROS during root hair development.

Figure 6.

Coregulation of SODs and RBOHs by protein‐protein interactions. (A, B) In silico modeling of RBOHC‐RACK1A‐FSD1 complex using AlphaFold2 Multimer as visualized in icn3d Structure Viewer (https://www.ncbi.nlm.nih.gov/Structure/icn3d/full.html). Visualisation of RACK1A‐RBOHC (A) and RACK1A‐FSD1 interface (B) in the complex. RACK1A structure is highlighted in blue in A and B. RBOHC is highlighted in green in A and grey in B. FSD1 is highlighted in grey in A and green in B. The protein‐protein interaction interfaces are depicted in yellow. [Color figure can be viewed at wileyonlinelibrary.com]

Another regulatory protein complex with the capability to impact both SODs and RBOHs is the G‐protein complex. According to high‐throughput Y2H results, FSD1 might also be involved in G‐protein signaling, as it was shown to interact with the GUANINE NUCLEOTIDE‐BINDING PROTEIN ALPHA‐1 SUBUNIT (GPA1), GUANINE NUCLEOTIDE‐BINDING PROTEIN SUBUNIT BETA (AGB1), and an effector of the beta subunit called ACIDOREDUCTONE DIOXYGENASE 2 (ARD2) (Klopffleisch et al. 2011). RACK1A interacts with regulator of G‐protein signaling 1 (AtRGS1), AGB1 and guanine nucleotide‐binding protein subunit gamma 1 and 2 (AGG1/2) (Olejnik et al. 2011). RBOHs are also regulated by protein‐protein interaction with the G‐protein complex (Zhang et al. 2021). GPA1, the subunit also interacting with SOD interact also with RBOHD during immune response (Xu et al. 2019). This means that potentially G‐protein complex might bring SODs, RACK and RBOHs together to control redox homeostasis during developmental processes.

RACK1A affects brassinosteroid (BR), ethylene and ABA signaling by interaction with critical regulatory proteins such as BRASSINAZOLE RESISTANT 1 (BZR1) (Li et al. 2023), ABSCISIC ACID INSENSITIVE 5 (ABI5) (Li et al. 2022; Li et al. ²⁰²⁴) and ETHYLENE‐INSENSITIVE 3 (EIN3) (Masood et al. 2023), thus impacting hypocotyl elongation, seed germination and post‐germinative growth and leaf senescence, respectively. During ABA signaling, RACK1A interacts with key components of the miRNA processing and effector complexes, SERRATE (SE) and ARGONAUTE 1 (AGO1), including the regulation of miR398, which directly affects CSD expression (Speth et al. 2013). Auxin induces the phosphorylation of RACK1A on Tyr248, leading to its homo‐dimerization promoting lateral root formation (Alshammari et al. 2021). Thus, RACK1A may control ROS levels in response to changes in endogenous phytohormone signaling.

An interesting link between SOD and NADPH oxidase was found in mammals. Mammalian SOD1 may regulate the Nox1 NADPH oxidase by direct interaction with Rho GTPase Rac1. The binding of SOD to Rac1 under reducing conditions leads to inhibition of GTP hydrolysis by Rac1. However, in oxidizing conditions, the SOD1‐Rac1 complex dissociates, allowing for GTP hydrolysis by Rac1. A point mutation in SOD1, connected with Amyotrophic lateral sclerosis, leads to O₂ ^•‐ overproduction by activating Rac1/Nox1 module (Harraz et al. 2008). Constitutive active Rac1 also has consequences on SOD1 (Pesaresi et al. 2011). Although interaction between RopGTPases and NADPH oxidases was reported also in plants (Wong et al. 2007), the Rop GTPase – SOD interaction remains to be functionally characterized. Thus, small RopGTPases might represent another protein potentially co‐regulating SODs and RBOHs (Figure 6B), although the SOD‐RopGTPase interaction was not identified in plants so far.

6. Conclusions

Current research has accumulated growing evidence about the spatial and temporal balance between O₂ ^•‐ and H₂O₂ during developmental processes. Therefore, understanding how O₂ ^•‐ production and scavenging are regulated spatially and temporally is a key focus.

Deeper insights into these aspects require detailed data on the spatial and temporal expression or enzymatic activation of ROS‐producing and scavenging enzymes. The emerging most critical questions include: (1) Which mechanisms coregulate O₂ ^•‐ production with decomposition? (2) By which mechanisms might mitochondrial, chloroplastic or peroxisomal O₂ ^•‐ transduce the signal to developmental processes? (3) Do SODs perform their roles beyond their O₂ ^•‐ decomposing activities?

To address these questions, it will be essential to advance the cell biology of antioxidant enzymes by applying high‐resolution microscopy together with precise markers of ROS and plant hormone localization and accumulation, including specialized biosensors (Rowe et al. 2023; Ugalde and Meyer ²⁰²⁵) and fluorescent probes (Kuběnová et al. 2022; Chen et al. ²⁰²⁴). For example, techniques of single‐cell microscopy might be feasible to observe in meristematic regions such as the SAM and RAM, using various SODs and RBOHs mutant backgrounds to dissect the precise regulation of hormonal production and ROS production and scavenging. Integrated approaches that combine molecular biology, microscopy, or proteomics are essential to precisely understand the mechanisms of ROS control. For example, proximity labelling, examined during phytohormone‐mediated developmental processes, to reveal the interacting partners of antioxidant enzymes might be effective in searching for their novel protein regulators. Similar approaches, combined with redox proteomics, may reveal novel redox‐regulated scaffold proteins that co‐regulate O₂ ^•‐ production and decomposition. The cooperation of chemical, cell biology, and molecular biology approaches will be beneficial in developing novel, more specific chemical probes or genetically encoded biosensors to differentiate ROS in individual cells or at the subcellular level. Such a complex, multidisciplinary approach may be beneficial for gaining more insights into the regulation of ROS in developmental processes. Such knowledge may represent a crucial foundation for future crop biotechnological advancements.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting Table 1: List of predicted and identified redox prosttranslational modifications of cysteines in the amino acid sequences of Arabidopsis and rice SODs and RBOH isoforms. as predicted by pCysMod database (http://pcysmod.omicsbio.info/). Redox modifications identified with FPR higher than 40 % were removed.

Supporting Table 2: List of identified prosttranslational modifications in the amino acid sequences of Arabidopsis and rice SODs and RBOH isoforms. as predicted by Plant PTM Viewer 2.0 database (https://www.psb.ugent.be/webtools/ptm-viewer/index.php).

Supporting Table 3: List of identified cis elements in promoter sequences of RBOH an SOD isoforms (2000 bp upstream of start codon) as identified by PLANTCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) database.

Data Availability Statement

The data that supports the findings of this study are available in the Supporting material of this article.