H₂O₂, NO, and H₂S networks during root development and signalling under physiological and challenging environments: Beneficial or toxic?

Abstract

Hydrogen peroxide (H₂O₂) is a reactive oxygen species (ROS) and a key modulator of the development and architecture of the root system under physiological and adverse environmental conditions. Nitric oxide (NO) and hydrogen sulphide (H₂S) also exert myriad functions on plant development and signalling. Accumulating pieces of evidence show that depending upon the dose and mode of applications, NO and H₂S can have synergistic or antagonistic actions in mediating H₂O₂ signalling during root development. Thus, H₂O₂‐NO‐H₂S crosstalk might essentially impart tolerance to elude oxidative stress in roots. Growth and proliferation of root apex involve crucial orchestration of NO and H₂S‐mediated ROS signalling which also comprise other components including mitogen‐activated protein kinase, cyclins, cyclin‐dependent kinases, respiratory burst oxidase homolog (RBOH), and Ca²⁺ flux. This assessment provides a comprehensive update on the cooperative roles of NO and H₂S in modulating H₂O₂ homoeostasis during root development, abiotic stress tolerance, and root‐microbe interaction. Furthermore, it also analyses the scopes of some fascinating future investigations associated with strigolactone and karrikins concerning H₂O₂‐NO‐H₂S crosstalk in plant roots.

Summary statement

• ‐NO and H₂S cooperative in the mechanism of modulating H₂O₂ homoeostasis in root development under normal and abiotic stress conditions.
• ‐Protein thiol‐based oxidative posttranslational modifications (oxiPTMs) are key elements in the coordination of root development.

1. INTRODUCTION

In the article “The ‘root‐brain’ hypothesis of Charles and Francis Darwin”, Baluška et al. (2009) elaborated the Charles Darwin's thoughts on root communications as “It is hardly an exaggeration to say that the tip of the radicle thus endowed [with sensitivity] and having the power of directing the movements of the adjoining parts, acts like the brain of one of the lower animals; the brain being seated within the anterior end of the body, receiving impressions from the sense‐organs, and directing the several movements”.

Considering this general idea, there are currently enough experimental data supporting that the variations in morphology and regulation of root architecture are orchestrated by the interplay of several signalling molecules which are in turn controlled during physiological and stressed environments (Meng et al., 2019; Motte et al., 2019; Su et al., 2017). ROS, particularly H₂O₂, are multitaskers in regulating root development especially meristem maintenance, root elongation, lateral root branching, adventitious rooting, endodermis formation, and differentiation of vascular tissue. H₂O₂ levels are crucially regulated in the meristematic and elongation zones of growing root apices, where nutrient stress, pH change, and various other environmental adversities regulate cell expansion and cell division in and around the quiescent centre (Eljebbawi et al., 2020; Francoz et al., 2019; Mangano et al., 2017). ROS‐mediated transcriptional regulation is essential for the transition of root tips from proliferative to the differentiating stages (Tsukagoshi et al., 2010). Among the numerous plant growth regulators associated with root development, during the last decade, two gasotransmitters, NO and H₂S, have gained relevant significance for their implications in root growth under physiological and stressful conditions (Kou et al., 2018; Li et al., 2016; J. Li et al., 2020; C. Li et al., 2020; Li et al., 2020a, 2020b; H. Li et al., 2021; Ma et al., 2020; Mishra et al., 2021; D. Piacentini, Della Rovere, et al., 2020; D. Piacentini, Corpas, et al., 2020; Zhou et al., 2018). Plant NO biosynthesis is commonly carried out by enzymatic and nonenzymatic pathways (Kolbert et al., 2019). Currently, nitrate reductase (NR) and NO‐like synthase activity are the major enzymatic sources of NO production in plants (Corpas et al., 2022a and references therein). The biosynthesis of H₂S in plants is prevalently accomplished by enzymatic reactions (Aroca et al., 2021; Gotor et al., 2015) involved in cysteine (Cys) metabolism which primarily comprise the activity of l‐Cys desulfhydrase, d‐Cys desulfhydrase, sulphite reductase, cyanoalanine synthase, and Cys synthase (González‐Gordo et al., 2020 and references therein).

In the last decade, the interest in ROS signalling during root development has been represented by around 1119 publications according to the PubMed database (Figure 1a), whereas NO and ROS interaction in root development, with 104 publications, has exhibited more than a 3‐fold increase in the last decade (Figure 1b). On the other hand, NO‐H₂S interactions during root development include around 55 publications. The interactions of H₂S‐ROS in root development represented by around 17 publications are included in Table 1. The associative interactions among NO, H₂S, and ROS in root development and physiology are also analyzed in eight investigations under normal or stressful environments (see Table 1).

Figure 1.

(a) Number of publications in the period of 2011–2021 representing reactive oxygen species (ROS) signalling in root development. (b) Venn diagram analysis of the number of publications on the different signal molecules, namely nitric oxide (NO), hydrogen sulphide (H₂S), and ROS, related to root development, found in the PubMed database in the same period 2011–2021. [Color figure can be viewed at wileyonlinelibrary.com]

Table 1.

Examples of signalling events associated with NO, H₂S, and H₂O₂ crosstalk in plant roots under physiological or abiotic stress

Plant species	Signalling events	Rooting response	References
Physiological condition
Solanum lycopersicum	H2S‐induced H2O2 production	Enhanced lateral root growth	Mei et al. (2017)
Solanum lycopersicum	NO‐induced H2S accumulation	Enhanced lateral root growth	Y.J. Li et al. (2014)
Vigna radiata	H2S functions as an upstream component to H2O2	Increased rate of radicle protrusion	Li and He (2015)
Arabidopsis	H2O2‐mediated MPK6 activity and NO generation	Promotion of lateral root growth in the mutants mpk6‐2 and mpk6‐3	Wang et al. (2010)
Tagetes erecta	Synergistic action of NO and H2O2; NO‐induced H2O2 generation	Promotion of adventitious rooting	Liao et al. (2009)
Dendranthema morifolium Beiguozhicun	Synergistic action of NO and H2O2; upreglation of polyphenol oxidase and IAa oxidase activity	Promotion of adventitious rooting	Liao et al. (2010)
Salinity
Cucumis sativus	Exogenous NaHS caused reduction in H2O2 accumulation	Promotes primary root growth, lateral root branching and root dry weight	Jiang et al. (2019)
Arabidopsis thaliana	H2S‐induced H2O2 production	Improved primary root growth	N. Li et al. (2014)
Bruguiera gymnorrhiza (L.) Savigny and Kandelia candel (L.) Druc	Exogenous NO and H2O2 regulate secondary active ion transport	Stress resilience, and ion homeostaisis	Y. Lu et al. (2013)
Arabidopsis thaliana	cGMP‐mediated H2O2 generation	Improves primary root growth and ion homeostaisis	J. Li et al. (2011); Li and Yia (2013)
Water stress
Zea mays	Triggers apoplastic H2O2 generation	Reduction in root apex cell division and reduced primary growth	Voothuluru et al. (2020)
Triticum aestivum	H2O2 and NO‐induced modulation of ABA sensitivity	Increases root growth	Habib et al. (2020); Kaur and Zhawar (2017)
Drought stress
Tagetes erecta	NO and H2O2‐mediated stress alleviation	Promotion of adventitious rooting	Liao et al. (2012)
Hypoxia stress
Pisum sativum	H2S‐induced reduction in H2O2	Inhibition of root tip death	Cheng et al. (2013)
Prunus persica L. Batsch	H2S‐induced reduction in H2O2	Inhibition of root tip death	Xiao et al. (2020)
H 2 S toxicity
Arabidopsis thaliana	RBOH‐mediated NOS‐like activity increases via MPK6 signalling	Inhibition of primary growth	Zhang et al. (2017)
Arsenic stress
Oryza sativa	NO‐induced ROS generation	Increases number of adventitious rooting and improves primary root length	Kushwaha et al. (2019)
Oryza sativa	NO‐induced reduction in H2O2 content	Amelioration of root growth inhibition	Singh et al. (2009)
Glycine max	NO and H2O2‐mediated antixoxidative defence	Promotion of root growth	Singh et al. (2020)
Metal stress (Cd, Al, Pb, Ni, Zn, and Cu)
Brassica campestris ssp. chinensis	H2‐water induced apoplastic H2O2 formation by RBOH activity	Increased primary root elongation and reduced Cd uptake	Wu, Huang et al. (2020)
Triticum aestivum	H2O2‐mediated downstream NO biosynthesis	Increased root length and enhanced antioxidative defence under Al stress	Sun et al. (2018)
Triticum aestivum	NO‐induced reduction in H2O2 content	Alleviation of radicle growth inhibition under Pb stress	Kaur et al. (2015); C. Kaya, Ashraf, et al. (2020)
Cucurbita pepo	NaHS (H2S)‐induced reduction in H2O2 content	Improved primary root growth under Ni stress	Valivand et al. (2019)
Triticum aestivum	NO and H2O2 mutually influence their biostnthesis	Inhibition of root growth under Zn stress	Duan et al. (2015); Karpets et al. (2015)
Matricaria chamomilla	Cu stress	Increased lignification in roots under Cu stress	Kováčik et al. (2010)
	NO and H2O2‐induced lignin metabolism
Antibiotic (oxytetracycline) toxicity
Solanum lycopersicum	Antibiotic (oxytetracycline) toxicity	Inhibition of primary root growth	Yu et al. (2017)
	Suppression of H2O2 and accumulation of NO
Analysis with oas‐a1.1 and osa‐a1.2 mutants (cysteine biosynthesis knock‐out)
Arabidopsis thaliana	Mutant analysis with oas‐a1.1 and osa‐a1.2 mutants (cysteine biosynthesis knock‐out). Reduced H2O2 production	Reduced primary root length	M.C. López‐Martín, Becana, et al. (2008)
Integrative roles of NO and H 2 S and ROS (stress conditions)
Capsicum lycopersicon L.	Exogenous NO regulates H2S‐dependent ROS generation	Modulates root growth and biomass (Cr stress)	Alamri et al. (2020)
Capsicum annuum L.	Endogenous NO and H2S levels regulate ROS content	Changes in root biomass (salinity and Fe‐deficiency)	C. Kaya, Higgs, et al. (2020)
Sesamum indicum	NO and H2S crosstalk determines ROS content	Regulates Pb accumulation in roots	Amooaghaie et al. (2017)
Zea mays	Combined effect of NO and H2S regulate ROS content	Improves radicle length in Cr stress	Kharbech et al. (2020)
Vigna radiata	Involvement of Ca2+ in NO and H2S interaction	Regulate root growth and ROS generation	Khan et al. (2020)
Pisum sativun	H2S regulates NO and ROS	Improves root biomass	Singh et al. (2015)
Zea mays	Combined effect of NO and H2S regulate ROS content	Promotes hypoxia tolerance	Peng et al. (2016)
Cucumis sativus	H2S‐NO‐MAPK signalling modulate ROS generation	Regulates root biomass and nitrate stress tolerance	Qi et al. (2019)

This review analyses the link between NO, and H₂S pathways in H₂O₂ signalling during root development and microbial interactions under physiological and stress conditions. Furthermore, it evaluates the plausible roles of strigolactones and karrikins in modulating H₂O₂‐NO‐H₂S crosstalk in plant roots.

2. NATURE OF INTERACTIONS OF NO, H₂S, AND H₂O₂: WHICH TAKES THE CENTRE STAGE?

Growing pieces of evidence suggest that NO and H₂S share a wide range of physical properties due to their possessing similarities or complementariness of physiological functions. However, NO and H₂S do not function independently but have substantial overlapping roles in plants. NO is a free radical that can produce a group of derived molecules called RNS. H₂S is chemically a weak acid in nature which can dissociate in aqueous solutions into hydrosulfide (HS^–) and sulphide (S₂ ^–) anions. It is remarkable to mention that H₂S can interact with other oxidants like H₂O₂, superoxide radical (O₂ ^•‐), the hydroxyl radical (^•OH), and peroxynitrite (ONOO^–) which, however, requires further clarification in the networks of plant metabolism (Filipovic et al., 2018). An example is the S‐nitrosothiol thionitrous acid (HSNO) as cellular redox regulation, which is generated by the interaction between NO and H₂S (Antoniou et al., 2020; Kashfi et al., 2015; Marcolongo et al., 2019).

Likewise, NO also interacts with H₂O₂ and other oxidants in plant cells. NO can chemically react with O₂ ^•‐ to form ONOO^‐ which can trigger protein nitration and loss of function of target proteins. Insights into the nature of the interaction between NO and ROS have been critically analyzed by several researchers in the recent past (Del Castello et al., 2019; Groß et al., 2013; Nicolas‐Francès et al., 2022; Scheler et al., 2013). NO–ROS interactions might exhibit synergistic and antagonistic interactions in plant metabolic pathways and physiological responses (Del Castello et al., 2019, Innocenti et al., 2007, Zhou et al., 2005). Reports in Arabidopsis reveal 612 proteins as common targets of persulfidation and S‐nitrosation (Aroca et al., 2018). For instance, catalase, APX, and RBOH are targets of both persulfidation and S‐nitrosation which indicate the common interface of NO and H₂S interaction in regulating ROS metabolism. Since various overlapping functions of NO and H₂S regulate H₂O₂ metabolism, MAPK signalling, and protein oxiPTMs in plant roots (Corpas et al., 2022b), it is difficult to generalize the complexities of interaction or decipher the central role of NO or H₂S in each signalling network. Both gasotransmitters share upstream or downstream positions in various signalling networks deciphered about normal or challenging environments. H₂O₂ can exert either beneficial or deleterious effects in plants subjected to variable environmental conditions. Metabolic pathways operating within plant organelles are modulated by various stress conditions which could generate uncontrolled H₂O₂ during oxidative stress (Feng et al., 2020; Smirnoff & Arnaud, 2019; Ugalde et al., 2021; Zhu et al., 2018). H₂O₂ acts as a local and long‐distance‐systemic signalling molecule in plant organs occurring in the range of 50–5.000 nM g⁻¹ FW (Fichman & Mittler, 2020; Fraudentali et al., 2020; Niu & Liao, 2016; Zhu et al., 2015). The direct interaction of NO and NO‐derived molecules can mediate protein posttranslational modifications (PTMs), mainly S‐nitrosation and tyrosine nitration, and modulate key enzymes of the ROS metabolism (Begara‐Morales et al., 2013a, 2013b; Chaki et al., 2015; Gupta et al., 2020; Kohli et al., 2019; Palma et al., 2020). Protein S‐nitrosation comprises the covalent attachment of an NO group to the thiol (–SH) side chain of Cys. It is reversible and allows the regulation of the function of target proteins. On the other hand, tyrosine nitration comprises the addition of a nitro group (–NO₂) to one of the two equivalent ortho carbons of the aromatic ring of Tyr residues. It is irreversible PTM, and its consequence might be either the gain or loss of function of the affected protein or even no effect; nevertheless, in higher plants, the most frequent effect is the loss of function of affected proteins (Begara‐Morales et al., 2016). Integration of the role of various transcription factors associated with ROS‐phytohormone signalling, cell cycle control, and root growth confirms that Arabidopsis superoxide‐RBOH C isozyme is involved in root hair growth (Foreman et al., 2003; Mangano et al., 2017; Tsukagoshi, 2016; Zhou et al., 2020). RBOH and auxin signalling mutants such as rbohH:rbohJ, iaa14/solitaryroot 1‐1 (slr1), rsl4‐1, rbohC, and rbohD are likely susceptible to various environmental stresses and exhibit pathogen sensitivity and disrupted polar growth of root hair and pollen tubes (Kaya et al., 2015; Krieger et al., 2016; Mangano et al., 2017; Orman‐Ligeza et al., 2016; Smirnoff & Arnaud, 2019; Takeda et al., 2008).

It is important to remark on the relevance of NO and H₂O₂ interplay at the level of their biosynthesis in roots and other plant organs during physiological and environmental stresses (Airaki et al., 2011, 2015; Suhel et al., 2020). Intriguing details remain unanswered for NO‐mediated regulation of H₂O₂ levels in plants. MAPK signalling regulates the interrelations between NO and H₂O₂ production (de Pinto et al., 2006; Delaunay et al., 2000; Jammes et al., 2009; Lin et al., 2012, 2019). For instance, biochemical and genetic analyses denote that MPK6 activity is essential for NO and H₂O₂ production during Arabidopsis root development (Wang et al., 2010). In certain cases, the oxidative pathway for NO generation in plants is known to regulate cellular ROS levels (Chaki et al., 2009). The l‐Arg‐dependent pathway of NO production seems to involve the production of certain polyamines namely spermidine, spermine, and putrescine (Groppa et al., 2008; Hasan et al., 2021; Tun et al., 2006). Interrelations among NO, polyamine, and ROS homoeostasis have been deciphered concerning root development, guard cell signalling, and pollen tube growth where polyamine levels regulate endogenous NO‐mediated enzymatic ROS metabolism (Agurla et al., 2018; Benkő et al., 2020; Groppa et al., 2008; Kaszler et al., 2021; Recalde et al., 2018; Szepesi et al., 2022). While polyamines are regulators of lateral root primordia in Arabidopsis (Kaszler et al., 2021), their levels are crucial for maintaining ROS–NO balance in salicylic acid‐induced root growth in tomato (Szepesi et al., 2022). Thus, it is likely that polyamines metabolism through polyamine oxidases regulates ROS levels (Benkő et al., 2022) but also NO (Corpas et al., 2022a; Tun et al., 2006).

H₂S can also regulate ROS metabolism by another oxiPTM‐designated persulfidation, a reversible protein modification that involves the interaction of a cysteinyl thiol (‐SH) group with H₂S to generate the corresponding persulfide (–SSH) (Corpas et al., 2022b). Key antioxidant enzymes such as ascorbate peroxidase (APX) or catalase, and the superoxide‐generating RBOH are targets of persulfidation but also their own H₂S‐generating l‐cysteine desulfhydrase (Aroca et al., 2015; Corpas et al., 2019; Fu et al., 2018; C. Li et al., 2020; Shen et al., 2020). Figure 2 summarizes enzymatic routes of NO, H₂O₂, and H₂S metabolism in plant cells. In response to diverse external stimuli, abiotic stress, and soil nitrate levels, plant roots alter NO production (Hossain et al., 2015; Planchet et al., 2005; Stöhr & Ullrich, 2002; Wany et al., 2018). The H₂O₂ production is primarily regulated by a complex network of enzymatic and nonenzymatic scavenging processes in the chloroplasts, mitochondria, peroxisomes, and plasma membrane (Corpas et al., 2020; Foyer & Hanke, 2022; Smirnoff & Arnaud, 2019). A significant amount of intracellular H₂O₂ is produced by the H₂O₂‐producing oxidase enzymes (glycolate oxidase, acyl‐CoA oxidase, polyamine oxidases, and sulphite oxidase) during photorespiration, β‐oxidation of fatty acids, polyamine, and sulphur metabolism in peroxisomes (Corpas and Barroso, 2014; Corpas et al., 2020). Our current understanding of the regulatory roles of H₂S in maintaining ROS homoeostasis in roots comes from studies on abiotic stresses such as nutrient deficiencies, salinity, drought, osmotic stress, heavy metal and metalloid stress (Corpas et al., 2019; Jia et al., 2015; Lisjak et al., 2010, 2013; Mei et al., 2017; Wang et al., 2012; Wu et al., 2021; Zhang et al., 2017). RBOH transcripts are upregulated by the exogenous H₂S application in plants (Mei et al., 2017). Transcriptomic analyses of the knockout mutants of O‐acetylserine(thiol) lyase isozymes support that the raised of Cys metabolism and subsequent H₂S formation are linked to ROS production (Aݩlvarez et al., 2010; M.C. López‐Martin, Becana, et al., 2008; M.C. López‐Martin, Romero, et al., 2008). F. Liu et al. (2020) demonstrated that the treatment with the H₂S donor sodium hydrosulfide increased the RBOH gene expression in cucumber seedlings and, consequently, enhanced H₂O₂ accumulation. Pharmacological evidence supports that H₂S acts as a downstream component of H₂O₂ in cells (Ma et al., 2018). On the contrary, the integration of pharmacological, physiological, and genetic approaches supports that H₂S‐mediated ROS production is accompanied by a concomitant NO accumulation in Arabidopsis roots, thus a high H₂S content inhibits root growth and also triggers ROS accumulation (Zhang et al., 2017).

Figure 2.

The main metabolic routes of NO, H₂O₂, and H₂S generation in higher plant cells. (a) Nitrate reductase (NR) and l‐arginine‐dependent nitric oxide synthase (NOS)‐like activity are the recognized major enzymatic NO sources in the different subcellular compartments (Astier et al., 2018; Corpas et al., 2022a; Z. Kolbert, Barroso, et al., 2019; Mohn et al., 2019). (b) H₂O₂ is mainly produced in the chloroplasts, mitochondria, peroxisomes, and plasma membrane, where a complex network of enzymatic and nonenzymatic scavenging systems regulates its homoeostasis (Corpas, 2015; Smirnoff & Arnaud, 2019). The H₂O₂ forming oxidase enzymes (glycolate oxidase, acyl‐CoA oxidase, sulphite oxidase) participate in photorespiration, fatty acid β‐oxidation, and sulphur metabolism in peroxisomes thus yielding a considerable amount of intracellular H₂O₂ (Corpas et al., 2020; del Rio & López‐Huertas, 2016). (c) The biosynthesis of H₂S in plants is prevalently accomplished by enzymatic reactions as part of sulphur and cysteine metabolism (Gotor et al., 2015) which primarily involve the activity of L/D‐DES, CAS, SAT, OAS‐TL, and SiR. ACO, acyl‐CoA oxidase. APS, adenosine 5′‐phosphosulfate; APR, APS reductase; CAS, cyanoalanine synthase; ETC, electron transport chain; GOX, glycolate oxidase; L/D‐DES, L/D‐cysteine desulfhydrase; OAS, O‐acetylserine; NR, nitrate reductase; NiR, nitrite reductase; RBOH, respiratory burst oxidase homologs; SAT, serine acetyltransferase; SiR, sulphite reductase. [Color figure can be viewed at wileyonlinelibrary.com]

NO and H₂S undergo mutual interaction with each other and S‐nitrosation, tyrosine nitration, and persulfidation are the major PTMs mediated by these two gasotransmitters (Corpas & Barroso, 2015) which can affect protein function. Superoxide dismutase (SOD), catalase, APX, glycolate oxidase, and RBOH are some of the key enzymes that regulate H₂O₂ homoeostasis in plant cells (Chapman et al., 2019). Interestingly, NO and H₂S can differentially modulate the activity of these enzymes thus regulating the levels of H₂O₂. For instance, APX is negatively regulated by tyrosine nitration but upregulated by S‐nitrosation and persulfidation (Aroca et al., 2015; Begara‐Morales et al., 2013a; Z. Kolbert, Molnï, et al., 2019; Z. Yang et al., 2015). Catalase is negatively regulated by these three types of PTMs mediated by NO and H₂S (C. Li et al., 2020; Palma et al., 2020). Furthermore, Arabidopsis RBOHD is inhibited by S‐nitrosation Cys 890 (Yun et al., 2011) whereas is stimulated by persulfidation in the same Cys890 (Shen et al., 2020) which is a good example of antagonistic effects on this O₂ ^•‐‐generating enzyme. These data support that NO acts upstream to H₂O₂ metabolism (Corpas et al., 2020). NO, H₂S and H₂O₂ are likely to diffuse from the organelles into the cytosol of root cells where they might interact with other signalling molecules. Higher levels of NO and H₂S might stimulate ROS outbursts causing oxidative stress.

Therefore, both NO and H₂S can modulate H₂O₂ levels at various sites of its formation by direct modification (PTMs) in the ROS‐generating enzymes, or indirectly by involving intermediate signalling components like MAPKs, CDKs, and cyclins. Future works can bring about the possibility of deciphering the complex interactions of NO, H₂S, and H₂O₂ in plants. For example, similar to animal cells, polysulfide (H₂S_n) formation is expected to result from NO and H₂S interactions in plant cells (Miyamoto et al., 2017). In line with reports from the animal system, persulfides, polysulfides, and thionitrites (Hosseininasab et al., 2021) should gain more importance in investigations of NO and H₂S interactions in plant metabolism.

3. INTERACTIONS OF NO, H₂S, AND H₂O₂ IN THE REGULATION OF ROOT DEVELOPMENT AND ARCHITECTURE

Roots are the primary sensing organs that usually grow and proliferate in soil. This results in various challenges and obstacles for researchers to analyze their growth and architectural changes. The regulation of root morphology is coordinated by the orchestration of the three zones, namely, the meristematic, elongation, and differentiation zones. The meristematic and elongation zones are more sensitive to external cues like nutrients, pH, water, and other abiotic and biotic factors. ROS exert regulatory functions as signalling molecules in shaping the root architecture where are implicated several transcription factors and gene networks in the regulation of root meristem activity, root hair growth, initiation and extension of lateral root primordia, and restructuring of cell wall architecture (Kumar et al., 2020). The transient signals of NO and ROS outbursts are regulated by interactions with H₂S. Thus, transcriptional and posttranslational changes such as oxiPTMs S‐sulfenylation, S‐glutathionylation, S‐nitrosation, persulfidation, S‐cyanylation, and S‐acylation can modulate the expression and activity of various proteins associated with auxin signalling, ROS homoeostasis, primary metabolism, cytoskeleton rearrangement, and other signalling components during root development and signalling (Corpas et al., 2022b).

3.1. ROS (H₂O₂) signalling and root apex perception under normal and stress conditions

Root apex signalling and its elongation is a dynamic development process accomplished by the constant generation of cells in the maturation region. The ROS levels can orchestrate various stages of the cell cycle in root cells which also correlate with the expression of cyclins and cyclin‐dependent kinases (Hamdoun et al., 2016; Ortiz‐Espin et al., 2017; Tamirisa et al., 2017; Tognetti et al., 2017).

PROHIBITIN 3 (PHB3) is responsible for the maintenance of the stem cell niche in the growing root apical meristem, where, ROS production is essentially regulated by nutrient availability (Zhou et al., 2020). Stem cell homoeostasis in the shoot and root apex regulates organogenesis in plants. In this context, the balance between NO and ROS levels is intricately associated with gene expressions in the shoot and root apical meristem (Wany et al., 2018). Double mutants of nia1nia2 deficient in NO content and NR activity show impaired root meristem development being accompanied by diminished transcript expression of WUS‐related homeobox 5 (L. Sanz, Fernández‒Marcos, et al., 2015). RETRADED ROOT GROWTH and PHB3 regulate root stem cell niche (Zhou et al., 2011), wherein, PHB3 is needed for ROS‐coupled‐NO generation in the root apex (Wang et al., 2010). Soil N content (especially nitrate) can control NR‐dependent NO production in roots (Planchet et al., 2005). Cell levels of NO can trigger cytokinin‐induced expression of the cell cycle gene CYCD3 that influences stem cell proliferation and its homoeostasis (Shen et al., 2013). Root NO can regulate H₂O₂ generation which maintains the balance between stem cell niche and cell proliferation in the elongation zone of roots (L. Sanz, Fernández‐Marcos, et al., 2015). Plants subjected to variable N nutrition from soil (nitrate, ammonium, microbial activity) are closely linked with NO‐ROS generation, that in turn, regulates stem cell homoeostasis in root and shoot apices (Wany et al., 2018).

ROS levels, therefore, regulate the balance between the differentiation and proliferation of cells in the root apical meristem (Eljebbawi et al., 2020; Kong et al., 2018; Livanos et al., 2012; Sundaravelpandian et al., 2013). Interestingly, in the junction of elongation and meristem zone, H₂O₂ turnover is precisely regulated by the expression of some peroxidase isozymes (PRX39, PRX40, PRX57, and PRX71) (Manzano et al., 2014; Raggi et al., 2015; Vijayakumar et al., 2016). The expansion of the differentiation zone is brought about by a higher H₂O₂ accumulation which results from the inhibition of peroxidases. Table 2 summarizes different redox components associated with H₂O₂ homoeostasis and root signalling in higher plants.

Table 2.

Main components associated with H₂O₂ homoeostasis and root signalling in Arabidopsis thaliana including some examples in rice (Oryza sativa L.).

Acronym (Uniprot)	Location	Role in root development and signalling	References
Respiratory burst oxidative homolog (RBOH)
RBOHF (AT1G64060)	Root endodermis, lateral roots, stem, seedling, leaf, and inflorescence	Regulates H2O2 levels in the maturation, elongation, and differentiating zones of root apes. Negatively regulates LRP formation and downregulated peroxidase activity	Lee et al. (2013); Y.J. Li et al. (2014)
RBOHD (AT5G47910)
RBOHC (AT5G51060)	Expressed in the transition zone, elongation zone and root tip.	Generates ROS to activate Ca2+ channels, facilitates elongation of root hair	Mangano et al. (2017)
RBOHE (AT1G19230)	Expressed in emergence point of lateral root, stem, leaf, and inflorescence	Regulates lateral root development	Orman‐Ligeza et al. (2016)
Apoplastic type‐III peroxidases
PRX72 (AT5G66390)	Endodermis and xylem tissue in roots	Regulate xylem formation and ligini formation in roots	Herrero et al. (2013); Zhao et al. (2013)
PRX17 (AT2G22420)	Endodermix and xylem of roots	Participates in liginin polymerization via the H2O2 signalling	Hoffmann et al. (2020)
PRX64 (AT5G42180)	Root endodermis	H2O2‐mediated monolignol oxidation and lignin formation	Lee et al. (2013)
PRX37 (AT4G08770)	Root endodermis and vascular bundles	H2O2‐mediated phenol crosslinking activity durig lignin formation. Responsive to low temperature	Pedreira et al. (2011)
PRX07 (AT1G30870)	Lateral root and root hairs. Positively upregulated in presence of light and low temperature	Regulate root hair formation, ROS balance and lateral root emergence	Manzano et al. (2014); Vijayakumar et al. (2016)
PRX57 (AT5G17820)
PRX01 (AT1G05240)	Root hair. Co‐expressed with glycoprotein and expansin in cell wall	Cell wall extension of root hairs	Mangano et al. (2017); Marzol et al. (2022)
PRX44 (AT4G26010)
PRX73 (AT5G67400)
H 2 O 2 sensor (HPCA1, hydrogen peroxide induced Ca 2+ increases 1)
HPCA1 (AT5G49760)	Plasma‐membrane localized, activated by H2O2, cysteine modification and autophosphorylation of HPCA‐1	LRR‐RK receptor‐mediated calcium sensing	Wu, Chi, et al. (2022)
H 2 O 2 regulators
Retarded root growth (RRG) (AT1G69380)	Root meristem, primary and lateral root tip, quiescent centre, root meristem and stele	Promotes cell division in root apical meristem, negatively regulates cell expansion in apex	Zhou et al. (2011)
Prohibitin 3 (PHB3) (AT5G40770)	Vascular bundles, stem, leaves, redox homoeostasis in mitochondria	Inhibits cell cycle and maintains stem cell niche in quiescent centre. Regulated by H2O2 levels	Kong et al. (2018)
Casparian strip membrane domain proteins (CASPs) CASP1 (At2G36100)	Root endodermis	Regulate casparian strip formation and liginin polymerization	Alassimone et al. (2016); Li et al. (2018a)
Like sex four 2 (LSF2) (AT3G10940)	Root hair	Regulate H2O2 levels in root hairs	Zhao et al. (2016)
Feronia (FER) (At3G51550)	Root hair, induced by brassinosteroids	Regulates ROS‐mediated root hair development and upstream to RBOH activity	Dong et al. (2019); Duan et al. (2010); Kim et al. (2021)
Root hair defective six like4 (RSL4) (At1G27740)	Root hair, leaf, flower, induced by jasmonates and low phosphate deficiency	Promotes root hair growth	Mangano et al. (2017)
Hypoxia responsive ERF gene ERF 4 (At3G15210)	Lateral root	Regulates lateral root emergence	Shukla et al. (2019)
UPBEAT1 (UPB1) (At2G47270)	Root vascular bundle, root hair and lateral root cap	ROS homoeostasis and cell differentiation in roots	Manzano et al. (2014)
H 2 O 2 metabolism
Superoxide dismutase SOD (Cu–Zn, Mn)	Chloroplast, mitochondria, peroxisome, cytosol	Produces H2O2	Chen et al. (2022); Shafi et al. (2015)
AT2G28190
Catalase	Peroxisome	Removes H2O2	Li et al. (2015); Yang et al. (2019)
CAT 2 (At4g35090)
CAT 1 (Os03g0131200
Ascorbate peroxidase APX3 (At4g35000)	Chloroplast and cytosol	Transforms H2O2 to H2O and DHA	Correa‐Aragunde et al. (2013); Xu et al. (2018)
APX2 (Os07g0694700)

Analyses of the activity of ROS‐generating enzymes combined with transcriptomic and proteomic data show that the elongation rate of root tips is controlled by the balance between the thickening and loosening of the cell wall, both of which are controlled by ROS homoeostasis (Francoz et al., 2015; Mabuchi et al., 2018; Mase & Tsukagoshi, 2021). However, the precise mechanism of coordination between cell wall stiffening and expansion mediated by ROS during root elongation remains unanswered. H₂O₂ production in the endodermis results in the increased activity of PRX64 which oxidizes monolignol thus resulting in lignification around the Casparian strips (Franke, 2015; Lee et al., 2013). Double mutants of atrbohD1/F1 and atrbohD2/F2 have led to the observations that RBOHF is responsible for O₂ ^•− formation in the sites of Casparian strip where the latter dismutase to form H₂O₂ by the SOD activity and both RBOH isozymes negatively regulate lateral root development (N. Li et al., 2014). Root hair formation is coordinated by the action of ROS homoeostasis and H₂O₂ signalling wherein, RBOHC‐mediated ROS generation in the apoplast is followed by peroxidase activity which activates MAPKs signalling in the cytoplasm (Foreman et al., 2003; Sundaravelpandian et al., 2013). Molecular analysis involving the transcriptional regulation of the transcription factor ROOT HAIR DEFECTIVE SIX‐LIKE4 confirms that RBOHC is responsible for Arabidopsis root hair formation, although RBOHH and RBOHJ are also responsible for a smaller extent (Mangano et al., 2017).

H₂O₂ modulates root architecture by regulating root length, root elongation, lateral root formation, and adventitious rooting (Hernández‐Barrera et al., 2015; Liao et al., 2009; Liszkay et al., 2004; Ma et al., 2014). Exogenous H₂O₂ triggers changes in membrane potential due to Ca²⁺ influx activity which is an early response associated with the epidermis of the root elongation zone in Arabidopsis (Demidchik et al., 2007). Apoplastic H₂O₂ enters into the cytoplasm of root cells via the aquaporins (Eljebbawi et al., 2020; Hachez et al., 2013; Tian et al., 2016). However, a recent report involving a forward genetic screening approach identifies HYDROGEN PEROXIDE‐INDUCED Ca²⁺ INCREASES 1 (HPCA1) as the first cell surface sensor of H₂O₂ (Wu, chi et al., 2020). The function of HPCA1 has been observed to be unique in plants and is represented by a leucine‐rich‐repeat‐receptor kinase (LRR‐RK) which transduces tissue‐ and organ‐specific H₂O₂ signals (Wu, Chi, et al., 2020).

The analysis of RBOHI expression indicates that repression of lateral root growth in Arabidopsis during drought stress is mediated by the increase of RBOH activity and reduction in IAA sensitivity (He et al., 2017). Probe‐based monitoring of H₂O₂ distribution confirms that aluminium stress induces termination in root growth which is associated with a severe decline in H₂O₂ levels in the elongation zone of Arabidopsis roots (Hernández‐Barrera et al., 2015). The elongation zone and root apex regions are more susceptible to aluminium stress in comparison with the maturity zone (Hernández‐Barrera et al., 2015). Aluminium stress results in changes in the contents of cell wall polysaccharides thus reducing the activity of diamine oxidase or RBOH which decreases the amount of H₂O₂ as a rapid response to aluminium stress. H₂O₂‐mediated oxidative stress sensing in roots of Arabidopsis involves intracellular sequestration of plasma membrane aquaporins (plasma membrane intrinsic proteins; PIP) (Luu & Maurel, 2013; Wudick et al., 2015). Implying fluorescence spectroscopic analysis and by monitoring AtPIP2;1 expression, Wudick et al. (2015) propose H₂O₂‐mediated reversible adjustment of membrane properties and aquaporin functioning in roots during oxidative stress. Confocal laser scanning microscopic analyses of H₂O₂ distribution in the elongation and differentiation zone of Arabidopsis root tips reveal its precise role in root development (Dunand et al., 2007). A predominant H₂O₂ accumulation was observed in the differentiation zone and the cell walls of root hairs. The application of an H₂O₂ scavenger resulted in the promotion of root elongation and inhibition of root hair proliferation. The distribution of H₂O₂ in various regions of the root apex correlates with the spatial distribution of peroxidase activity.

3.2. NO modulates the H₂O₂ homoeostasis during root development

The association of NO and ROS in plant roots has been observed in various signalling pathways related to phytohormones and nutrient‐sensing under normal and challenging environments (Kushwaha et al., 2019; Szepesi et al., 2022; Verma et al., 2013; Zhang et al., 2017). Biochemical and genetic analyses prove that NO and H₂O₂ undergo transient changes in their endogenous levels which are finely tuned to various developmental, and environmental changes associated with root growth (Castillo et al., 2015; Kushwaha et al., 2019; Mukherjee & Corpas, 2020; D. Piacentini, Della Rovere, et al., 2020; Piacentini et al., 2021; Raya‐González et al., 2019; Sanz et al., 2015; Sun et al., 2018; Szepesi et al., 2022; Wei et al., 2018). Among the various signalling molecules associated with NO, auxin, ethylene, and brassinosteroids are known to be involved in NO‐associated ROS signalling pathways during root growth and development (Barrera‐Ortiz et al., 2018; Y. Fu, et al., 2019; Jacobsen et al., 2021; Liu et al., 2017, 2022; Raya‐González et al., 2019). The interaction of NO and auxin during root development involves the coordination of redox homoeostasis and the modulation of various redox regulatory proteins. Pharmacological studies demonstrate that NO exhibits auxin‐like action in regulating adventitious rooting‐ mediated by the action of NO donors such as sodium nitroprusside or S‐nitroso‐N‐acetyl penicillamine when applied to Cucumis sativus hypocotyls (Pagnussat et al., 2002).

Experimental data indicate that IAA, NO, and H₂O₂ function through canonical signalling pathways thus bringing about the rooting responses (Table 3). NO and H₂O₂ likely exhibit both independent and synergistic relations in bringing about rooting responses in plants. However, during auxin‐induced rooting NO–H₂O₂ interaction accompanied by cGMP signalling function as downstream components to auxin, wherein higher auxin and NO concentrations appear inhibitory to root growth (Mukherjee & Corpas, 2020, Raya‐González et al., 2019).

Table 3.

Root proteins identified to be potential targets of different oxiPTMS mediated by NO (S‐nitrosation), H₂O₂ (S‐sulfenylation), or H₂S (persulfidation) and its implication in different functional categories

Protein name	UniProt number	oxiPTMS	References
Root development, root hair formation and regulation of RSA
Vascular‐related nac‐domain7	Q9C8W9	S‐nitrosation	Kawabe et al. (2018)
Like sex four 2 (LSF2)	Q9SRK5	S‐sulfenylation	Huang et al. (2019)
Respiratory burst oxidase homolog protein D (RBOHD)	Q9FIJ0	Persulfidation	Jurado‐Flores et al. (2021); Mei et al. (2017); Shen et al. (2020)
Mod 1 (NAD(P)‐binding Rossmann‐fold superfamily protein	Q9SLA8	S‐nitrosation S‐sulfenylation	Hu et al. (2015); Huang et al. (2019)
Hydrogen peroxide sensor Hpca1	Q8GZ99	Predicted S‐sulfenylation	Eljebbawi et al. (2020
Pectinesterase	Q8GXA1	S‐nitrosation	Jain et al. (2018); Pan et al. (2021)
Xyloglucan‐endotrans‐glucosylase/hydrolase 2‐like	Q38909	S‐nitrosation	Pan et al. (2021)
TRANSPORT INHIBITOR RESPONSE 1	Q570C0	S‐nitrosation	Terrile et al. (2012)
Protein kinase superfamily protein SNF1	Q38997	S‐nitrosation S‐sulfenylation	Hu et al. (2015); Huang et al. (2019)
Auxin metabolism, transport and signalling
IBA ‐ specific acyl acid amido synthetase (GH3 family)	Q8GZ29	S‐sulfenylation Persulfidation	Huang et al. (2019); Jurado‐Flores et al. (2021)
DFL1 (DWARF IN LIGHT 1) (indole‐3‐acetic acid amido synthetase‐ GH3)	Q9LSQ4	Persulfidation	Jurado‐Flores et al. (2021)
WES1 (indole‐3‐acetic acid amido synthetase)	O81829	Persulfidation	Jurado‐Flores et al. (2021)
Auxin‐responsive family protein	Q9LSE7	Persulfidation	Jurado‐Flores et al. (2021)
ABP1 (ENDOPLASMIC RETICULUM AUXIN BINDING PROTEIN 1)	P33487	Persulfidation	Jurado‐Flores et al. (2021)
AXR1 (AUXIN RESISTANT 1)	Q96247	Persulfidation	Jurado‐Flores et al. (2021)
IAA‐Ala conjugate hydrolase/metallopeptidase	D7KIJ7	Persulfidation	Jurado‐Flores et al. (2021)
IAA‐Leu conjugate hydrolase/IAA‐Phe conjugate hydrolase/metallopeptidase	P54968	Persulfidation	Jurado‐Flores et al. (2021)
Indole‐3‐acetonitrile nitrilase	P32962	Persulfidation	Jurado‐Flores et al. (2021)
E3 ubiquitin ligase Auxi signalling	Q9C895	S‐nitrosation S‐sulfenylation	Huang et al. (2019); Terrile et al. (2022)
AUXIN RESISTANT 6 (component of ubiquitin 3 ligase)	Q9FZ33	S‐nitrosation S‐sulfenylation	Hu et al. (2015); Huang et al. (2019); Wei et al. (2020)
TSB 1 (tryptophan synthase beta‐subunit 1) Auxin precursor	P14671	S‐sulfenylation	Huang et al. (2019), Wei et al. (2020)
Other phytohormone signalling
Gibberellin‐regulated family protein	Q9LFR3	Persulfidation	Jurado‐Flores et al. (2021)
Cytokinin dehydrogenase	Q9FUJ2	Persulfidation	Jurado‐Flores et al. (2021)
Histidine‐containing phosphotransmitter 1 (AHP1) cytokinin signalling	Q9ZNV9	S‐sulfenylation	Parí et al. (2013)
Ethylene‐responsive protein, putative	Q84TF6	Persulfidation	Jurado‐Flores et al. (2021)
1‐aminocyclopropane‐1‐carboxylate oxidase, putative	O65378	Persulfidation	Jurado‐Flores et al. (2021)
Abscisic aldehyde oxidase	Q7G9P4	Persulfidation	Jia et al. (2018); Jurado‐Flores et al. (2021)
Open stomata 1 (OST1)	Q940H6	Persulfidation	Chen et al. (2020)
MYB 30 transcription factor for brassinosteroid signalling	Q9SCU7	S‐nitrosation	Tavares et al. (2014)
Primary metabolism
Aconitate hydratase	Q42560	S‐nitrosation	Jain et al. (2018)
		S‐sulfenylation	Hu et al. (2015); Wei et al. (2020)
Alcohol dehydrogenase 1	P14673	S‐nitrosation	Dumont et al. (2018); Huang et al. (2019); Jain et al. (2018)
		S‐sulfenylation
Glyceraldehyde‐3‐phosphate dehydrogenase	P25861	S‐nitrosation	Jain et al. (2018)
Isocitrate dehydrogenase	P50217	S‐nitrosation	Jain et al. (2018)
Malate dehydrogenase	O48905	S‐nitrosation	Jain et al. (2018); Pan et al. (2021); Huang et al. (2018)
		S‐sulfenylation
Malate synthase	P08216	S‐nitrosation	Jain et al. (2018)
NADP‐dependent malic enzyme	Q9XGZ0	S‐nitrosation	Hu et al. (2015); Pan et al. (2021)
ATP‐citrate synthase alpha chain protein 1	Q9FGX1	S‐nitrosation S‐sulfenylation	Pan et al. (2021); Wei et al. (2020)
Oxygen evolving enhancer protein 1	P85194	S‐nitrosation	Jain et al. (2018)
Phosphoenolpyruvate carboxykinase 6	P42066	S‐nitrosation	Jain et al. (2018)
Ribulose bisphosphate carboxylase large chain	P45738	S‐nitrosation	Jain et al. (2018)
Ribulose bisphosphate carboxylase small chain	P08705	S‐nitrosation	Abat and Deswal (2009); Jain et al. (2018)
Sedoheptulose‐1,7‐bisphosphatase	P46285	S‐nitrosation	Jain et al. (2018)
Triosephosphate isomerase	P48493	S‐nitrosation	Jain et al. (2018)
UDP‐glucose:4‐aminobenzoate acylglucosyltransferase	O22822	Persulfidation	Jurado‐Flores et al. (2021)
2‐oxoglutarate‐dependent dioxygenase	P93824	Persulfidation	Jurado‐Flores et al. (2021)
UDP‐glycosyltransferase	Q9FIA0	Persulfidation	Jurado‐Flores et al. (2021)
Adenosyl‐homocysteinase	P32112	S‐nitrosation	Jain et al. (2018)
S‐adenosylmethionine synthetase	Q9LUT2	S‐nitrosation S‐sulfenylation Persulfidation	Jurado‐Flores et al. (2021); Lindermayr et al. (2005); Wei et al. (2020)
S‐nitrosoglutathione reductase (GSNOR4)	Q96533	S‐nitrosation S‐sulfenylation	Huang et al. (2019); Matamoros et al. (2020); Pan et al. (2021); Ventimiglia and Mutus (2020)
Iron ion binding/oxidoreductase	P92949	Persulfidation	Jurado‐Flores et al. (2021)
Phospholipase D alpha 1	Q43007	S‐nitrosation	Jain et al. (2018)
Lipoxygenase LOX1	Q06327	Persulfidation	Jurado‐Flores et al. (2021)
d‐cysteine‐desulfhydrase	F4HYF3	Persulfidation	Jurado‐Flores et al. (2021)
UDP‐glucose 6‐dehydrogenase 1	Q9FZE1	S‐nitrosation	Jain et al. (2018)
ROS scavenging enzymes
Dehydroascorbate reductase (DHAR)	Q9FWR4	S‐nitrosation	Fares et al. (2011)
CATALASE 1	Q96528	S‐nitrosation S‐sulfenylation	Hu et al. (2015); Jain et al. (2018); Wei et al. (2020)
2‐Cys peroxiredoxin	Q9C5R8	S‐nitrosation S‐sulfenylation	Jain et al. (2018); Wei et al. (2020)
APX	Q05431	S‐nitrosation S‐sulfenylation	Huang et al. (2019); Jain et al. (2018); Wei et al. (2020)
MDAR6	B9DGR6	S‐nitrosation S‐sulfenylation	Hu et al. (2015); Huang et al. (2019); Jain et al. (2018)
Ascorbate peroxidase 1	Q05431	S‐sulfenylation Persulfidation	Fares et al. (2014); Correa‐Aragunde et al. (2013); Wei et al. (2020)
Cell signalling, stress tolerance and miscellaneous
Chaperone protein ClpB1	P42730	S‐nitrosation S‐sulfenylation	Huang et al. (2019); Jain et al. (2018)
Endoplasmin Homolog	P35016	S‐nitrosation	Jain et al. (2018)
Heat shock protein 90‐1	P27323	S‐sulfenylation	Jain et al. (2018); Wei et al. (2020)
HSP 70	Q8GUM2	S‐nitrosation S‐sulfenylation	Fares et al. (2011); Pan et al. (2021); Wei et al. (2020); Wei et al. (2020)
Annexin D1	Q9SYT0	S‐nitrosation	Jain et al. (2018)
40S ribosomal protein S3a	P49198	S‐nitrosation	Jain et al. (2018)
14‐3‐3‐like protein	O65352	S‐nitrosation	Jain et al. (2018)
Elongation factor 2	Q9ASR1	S‐nitrosation	Jain et al. (2018)
ATP synthase subunit alpha	P18260	S‐nitrosation	Jain et al. (2018)
ATP synthase Subunit β	Q9FT52	S‐nitrosation	Pan et al. (2021)
Aquaporin PIP1‐3	Q08733	S‐nitrosation	Jain et al. (2018)
Jacalin lectin family protein	O04314	Persulfidation	Jurado‐Flores et al. (2021)
Actin‐2	Q96292	S‐nitrosation S‐sulfenylation	Jain et al. (2018); Puyaubert et al. (2014); Wei et al. (2020)
Tubulin alpha‐2 chain	Q6VAG0	S‐nitrosation	Jain et al. (2018)
Calmodulin	P04464	S‐nitrosation	Jain et al. (2018); Pan et al. (2021)
Signal recognition particle 19 kDa protein, putative	Q943Z6	Persulfidation	Jurado‐Flores et al. (2021)
Myosin heavy chain‐related protein	Q9FHD1	Persulfidation	Jurado‐Flores et al. (2021)
Calreticulin	Q38858	Persulfidation	Jurado‐Flores et al. (2021)
Serine/threonine kinase target of rapamycin	Q39030	Persulfidation	Jurado‐Flores et al. (2021)
Methionine‐adenosyltransferase 3	Q9SJL8	S‐nitrosation S‐sulfenylation	Hu et al. (2015); Wei et al. (2020)
Tetratricopeptide repeat (TPR)‐like superfamily protein HSP 90	Q940R2	S‐sulfenylation	Wei et al. (2020)
NPR1	P93002	S‐nitrosation	Tada et al. (2008)

Abbreviation: RSA, root system architecture.

Among the various signalling molecules participating in NO and H₂O₂‐mediated pathways, cGMP, MPK6, and RBOH contribute to signalling events accompanying root development (Bai et al., 2012; Han et al., 2015; Mhamdi and Van Breusegem, 2018; Li & Xue, 2010; J. Li et al., 2011; Z.G. Li et al., 2013; Pagnussat et al., 2004; Pagnussat et al., 2003; Raya‐González et al., 2019; Singh et al., 2021). NO functions upstream to exert precise control over H₂O₂ homoeostasis by modulating the activity of peroxidase, catalase, APX, and SOD in plant roots subjected to adverse conditions (He et al., 2019; Z. Kolbert, Molnï, et al., 2019; Kushwaha et al., 2019; Singh et al., 2021). The positive role of NO on H₂O₂ catabolism by APX is through gene expression analysis of peanut plants exhibiting Al‐induced apoptosis (He et al., 2019). The authors report increased mitochondrial ROS generation accompanied by a higher ratio of SOD/APX, which indicates the accumulation of H₂O₂ in root tips subjected to aluminium stress. Exogenous NO priming alters H₂O₂ levels by reducing the ratio of SOD/APX thus decreasing apoptosis. 2‐D proteomic detection and activity analysis of recombinant APX show that auxin and NO‐mediated signalling induces NTR‐TRX‐based denitrosation of cytosolic APX in Arabidopsis which results in inhibition of APX activity and higher H₂O₂ formation accompanying lateral root formation (Correa‐Aragunde et al., 2015; Correa‐Aragunde et al., 2013).

The restoration of the redox milieu brought about by the regulatory antioxidant enzymes (SOD, APX, or catalase) provides clues to the fact that H₂O₂ has a dual role in the promotion and inhibition of rooting response which might vary in different plant species according to the treatment, soil regimes, physiological or stressed environment.

3.3. Can H₂S mimic the NO–H₂O₂ signalling during root development?

Plants are also exposed to external sources of H₂S which might act as a signal or growth‐inhibitory gaseous molecule at toxic extra‐physiological concentrations (Lamers et al., 2013; Liu et al., 2021). The crosstalk among NO, H₂S, and ROS provides us with an account of similarities in the function of the two gasotransmitters which influence ROS metabolizing enzymes and regulate H₂S homoeostasis in plant organs.

Similar to NO, H₂S regulates lateral root development, the application of 1 mM sodium hydrosulfide on tomato seedlings resulted in an upregulation of RBOH1 transcript thus resulting in H₂O₂ accumulation (Mei et al., 2017). Application of H₂O₂ scavengers revealed that H₂O₂ accumulation is crucial for lateral root primordia initiation and its elongation which is also accompanied by the regulation of cell cycle‐specific cyclins and cyclin‐dependent kinases (Eljebbawi et al., 2020). Contrastingly, in Arabidopsis seedlings, the application of sodium hydrosulfide in the range of 200 μM to 800 μM inhibited root growth (Zhang et al., 2017). The toxic concentration of H₂S is known to repress primary growth in Arabidopsis by mediating signal transduction pathways associated with ROS accumulation, activation of MITOGEN‐ACTIVATED PROTEIN KINASE 6 (MPK6), and NO accumulation (Zhang et al., 2017). Functional analysis using the mutants for RBOH (rbohD/F) and an NO low content (noa1) revealed that NO‐ROS crosstalk is essential for mediating the H₂S effects. Thus, ROS production and MPK6 activity appear in the centre place of NO–H₂S crosstalk and regulation of auxin distribution during primary root growth in Arabidopsis.

Overexpression of the L‐Cys desulfhydrase (LCD) gene triggers that the H₂S‐mediated persulfidation (Cys‐287) of actin 2 in Arabidopsis resulting in F‐actin bundle depolymerization thus leading to the inhibition of root hair growth (Li et al., 2018). H₂S also mediated an increase in SOD activity and H₂O₂ accumulation during the root development of strawberries (Hu et al., 2020). Pharmacological analysis during the proliferation and growth of root hairs in Linum album, reveals that exogenous H₂S promotes the accumulation of endogenous H₂O₂, H₂S, and NO in roots (Fakhari et al., 2019). This crosstalk among H₂S, NO, and H₂O₂ was accompanied by enhanced lignin biosynthesis in the roots.

Accordingly, H₂S and NO mostly imply similar molecules (MPK6, auxin, brassinosteroids, and RBOH) as intermediate components in H₂O₂ signalling during root development. H₂S and NO have a mutual influence on each other in regulating their biosynthesis and therefore, modulate H₂O₂ levels in roots. Figure 3 depicts various events (Boxes 1–7) associated with the regulation of ROS generation which in turn is associated with the genetic control of root proliferation, expansion, lateral root primordia initiation, and maintenance of stem cell niche. Several peroxidase isozymes are important regulators of ROS generation associated with lignification and cell wall extension in the lateral root primordial region (Böhm et al., 2010). Moreover, it would be worthwhile to decipher any possible roles of strigolactones, karrikins, and microbial metabolites (homoserine lactones) in regulating one or more routes of NO‐H₂S‐ROS signalling in roots (Kong et al., 2017; X. Zhang et al., 2020). Considering the similarities in the mode of action of NO and H₂S (oxiPTMs, MAPK signalling, and phytohormone crosstalk) and their interaction with ROS signalling it is imperative to understand that H₂S could also exert NO‐like action on the regulation of ROS generation thus modulating root development.

Figure 3.

Interactive roles of NO, H₂S, and H₂O₂ in the regulation of root development and signalling during normal and abiotic stress conditions. The root apex involves intimate coordination among the three signalling molecules NO, H₂S, and H₂O₂ in the meristematic zone (MZ), elongation zone (EZ), differentiating zone (DZ), and root cap (RC). Box 1, H₂S and NO jointly regulate the activity of key H₂O₂‐regulatory enzymes namely‐ superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) by posttranslational modifications which regulate H₂O₂ levels in the differentiating region of growing roots. Box 2, A paucity of information exists on the effects of NO and H₂S in regulating the activity of H₂O₂ sensor ‐ HYDROGEN PEROXIDE‐INDUCED Ca²⁺ INCREASES 1 (HPCA1) during root development. Box 3, PHB3 (PROHIBITIN 3) controls the stem cell niche at the quiescent centre (QC). H₂O₂ regulates the expression of PHB3 which crucially coordinates the balance between cell proliferation and cell expansion in the root apex during normal and adverse environmental conditions. Box 4, H₂S‐H₂O₂ crosstalk during the proliferation of root hairs, where H₂S‐mediated persulfidation of actin 2 at the Cys residues initiates H₂O₂ formation thus leading to root hair (RH) differentiation. Box 5, RBOHH and RBOHC are primarily associated with lateral root primordia (LRP) formation and lateral root growth where H₂O₂ production in the apoplast triggers peroxidase (PRX) activity. Apoplastic H₂O₂ in the LRP region might diffuse in the root cell cytosol and trigger the expression of cell division‐associated proteins namely cyclins (CYC) and cyclin‐dependent kinases (CDKs). Peroxidase‐mediated upregulation of extensins, pectin methyl esterase (PME), and polygalactouronase (PGE) promote cell elongation in the LRP region. Box 6, the transitional zone between MZ and EZ contains a higher expression of RBOH transcripts mediated by H₂S which in turn produces H₂O₂ followed by active expression of peroxidase genes (PRX 39, 40, and 57). This results in control of lignification. Box 7, NO‐H₂O₂ interaction includes crosstalk of NO, NO synthase‐like (NOS), N‐nitrosomelatonin (NOmela), and other RNS with H₂O₂ which also involve regulation of cGMP, IAA oxidase, MPK6, and polyphenol oxidase activity in the meristematic zone. [Color figure can be viewed at wileyonlinelibrary.com]

3.4. NO, H₂O₂ and H₂S‐mediated oxiPTMs regulate auxin signalling and metabolism accompanying the restructuration of root architecture under physiological and adverse conditions

The protein oxiPTMs (thiol‐based oxidative posttranslational modifications) are determined by the cellular location or environment of the susceptible target protein, for example, if it is a membrane protein or a soluble one (Corpas & Barroso, 2015; Zhang et al., 2021). Thiol groups in proteins typically exhibit a dissociation constant (pKa) in the physiological pH range of 7.0–7.4. Particularly, under stressful environments, various oxidant molecules trigger cell signalling associated with oxiPTMs which in turn modulate the activity and structure of numerous proteins (Castro et al., 2021). Protein thiol groups can undergo deprotonation to form negatively charged thiolate residues (Cys‐S⁻). Whereas oxidation of the thiol groups results in the formation of thiyl radicals (Cys‐S^•) and sulfenic acids (Cys‐SOH) (Turell et al., 2020). Sulfenic acid residues can be converted to sulfinic acid (‐SO₂H) followed by its irreversible conversion into sulphonic acid (‐SO₃H). NO, H₂S, and H₂O₂ mediate oxiPTMs such as S‐nitrosation, persulfidation, and S‐sulfenylation, respectively. S‐nitrosation involves a reversible covalent reaction mediated by NO or S‐nitrosothiols such as nitrosoglutathione formed by the chemical interaction between reduced glutathione and NO (Corpas & Barroso, 2015). Likewise, S‐sulfenylation (a reaction of H₂O₂ with Cys to form Cys sulfenic acid) has been studied by implying different probes which have led to the identification of up to 1,394 proteins in Arabidopsis cell cultures subjected to H₂O₂ treatment (Akter et al., 2015; De Smet et al., 2019; L. Fu et al., 2019; Huang et al., 2019; Waszczak et al., 2014; Wei et al., 2020). Persulfidation involves interaction between the thiol group of Cys residue and H₂S (Aroca et al. 2018; Wang et al., 2021) and seems to exert a more significant role in comparison with other oxiPTMs. In an attempt to search for root proteins as potential targets of NO, H₂O₂, and H₂S‐mediated oxiPTMs, we obtained information from the Uniprot database and available literature in the PubMed database. Using the PTM detector web tool “Plant PTM viewer” (https://www.psb.ugent.be/webtools/ptm-viewer/protein.php), we identified various root proteins as potential targets for the three types of oxiPTMs. A representative number of 85 identified root proteins (Table 3) were divided into various functional categories namely root development, auxin homoeostasis, another phytohormone signalling, primary metabolism, ROS scavenging, and stress‐related signalling. Among the identified 85 proteins, S‐nitrosation was observed in 50 proteins, persulfidation in 29 proteins, and S‐sulfenylation in 26 proteins. In this list, around 22 proteins were found to be targets of more than one oxiPTM. Some of the major proteins associated with root development, cell wall elongation, and ROS sensing involve Vascular‐related NAC‐domain7 (VND7), like sex four 2 (LSF2) pectinesterase, RBOHD, Actin2, and Hpca 1 (Table 3). S‐sulfenylation of proteins has been known to be associated with ACT2 depolymerization thus causing stunted root growth in Arabidopsis (Li et al., 2018). The role of VND7 is crucial for regulating vessel differentiation (xylogenesis) in roots (Yamaguchi et al., 2008). Similarly, LSF2 regulates root growth by regulating ROS homoeostasis and H₂O₂ generation in Arabidopsis (Zhao et al., 2016).

Auxin signalling components regulate root growth, meristem activity, and root differentiation in plants subjected to diverse environmental conditions. However, our current understanding of root phenotypic plasticity in response to environmental cues needs further attention for the control of auxin signalling (Parveen et al., 2022). The TIR1/AFB‐Aux/IAA‐ARF system of auxin‐dependent transcriptional regulation controls root development in response to spatial‐temporal auxin gradient (Morffy & Strader, 2021). Thus, auxin transport and its homoeostasis are crucial in mediating root meristem activity and root differentiation. Transcription factors like PLETHORA and the homeodomain transcription factor WUSCHEL RELATED HOMEOBOX 5 are needed for stem cell organization, auxin biosynthesis, and its transport in the root tips. Similarly, helix‐loop‐helix transcription factor ROOT HAIR DEFECTIVE 6 LIKE 4 mediates root hair differentiation through PIN/AUX1 signalling component. Although most of the components of the auxin signalling pathway are regulated by phosphorylation, certain proteins also exhibit persulfidation, while some of them are potential targets of S‐sulfenylation and S‐nitrosation (Hu et al., 2015; Huang et al., 2019; Jurado‐Flores et al., 2021; Terrile et al., 2022). Interestingly, proteins associated with auxin conjugation are mainly regulated by persulfidation in Arabidopsis roots under N starvation (Jurado‐Flores et al., 2021). TSB 1 (auxin precursor biosynthesis), Auxin resistant 6, and E3 ubiquitin ligase are regulated by both S‐sulfenylation and S‐nitrosation. Certain components of auxin signalling like ABP1 and DFL1 (DWARF IN LIGHT 1) exert photo‐modulatory regulation of lateral root formation and primary root growth (Nakazawa et al., 2001). Auxin resistance 6 protein is required for auxin perception and mediates the reshaping of root architecture under nutrient starvation (Williamson et al., 2001). Thus, NO‐, H₂O₂‐, and H₂S‐mediated oxiPTMs seem to regulate auxin signalling through its biosynthesis, conjugation, and subcellular sequestration in roots.

Jain et al. (2018) have identified several S‐nitrosated proteins in sunflower seedlings subjected to NaCl stress. The proteins were functionally categorized into primary metabolism, protein folding, cell trafficking, cytoskeleton elements, and other regulatory proteins. Table 3 represents some of the major proteins of primary metabolism, where S‐nitrosation seems to regulate carbohydrate metabolism in roots which might regulate metabolite partitioning in response to normal or adverse environmental conditions. Certain metabolic proteins like aconitate hydratase, alcohol dehydrogenase, malate dehydrogenase, UDP‐glycosyltransferase, lipoxygenase, and GSNO reductase are regulated by S‐sulfenylation and persulfidation in roots. Interestingly, the H₂S‐generating enzyme D‐Cys desulfhydrase is regulated by persulfidation during N stress (Jurado‐Flores et al., 2021). ROS scavenging enzymes in roots seem to be mostly regulated by S‐nitrosation and S‐sulfenylation. A significant number of identified root proteins belonging to cell trafficking, autophagy, protein folding, membrane transport, and signalling in roots also undergo modifications imposed by NO, H₂O₂, and H₂S (Aller & Meyer, 2013). Our current understanding of these oxiPTMs seems to exert overlapping effects on the regulation of root development, ROS homoeostasis, subcellular signalling, and primary metabolism. Interestingly, adverse conditions like N‐starvation or salinity stress also significantly induce persulfidation and S‐nitrosation of root proteins associated with auxin signalling, primary metabolism, and stress tolerance (Jurado‐Flores et al., 2021; Tanou et al., 2012). Figure 4 represents some of the important root regulatory proteins of auxin signalling, subcellular recognition, and conjugation modulated by the oxiPTMs. Certain proteins associated with lateral root primordia initiation, xylogenesis, and root hair elongation (Lsf2‐ Like sex four 2; Vnd7‐ Vascular‐related NAC‐domain7) are also regulated by oxiPTMs. Interestingly, NO, H₂O₂, and H₂S exert their effects on pectin methylesterase or xyloglucan endotransglycosylase activities which in turn control cell wall elasticity in the elongation zone. Similarly, the activity of RBOHD is regulated by persulfidation which functions in controlling the spatial distribution of ROS in the transition of the apex and meristematic zone. Apart from the regulation of auxin signalling and root architecture, physiological changes associated with salinity, nitrate stress, or oxidative burst (H₂O₂) have led to the identification of oxiPTMs in proteins functioning in starch biosynthesis, Calvin cycle, membrane lipid metabolism, and H₂S biosynthesis. The root apex is sensitive to environmental adversities which result in a transient change in NO/H₂O₂ levels tuned with H₂S metabolism. This results in the regulation of ROS‐scavenging enzyme activity such as APX, catalase, or SOD. Cell messenger proteins like 14‐3‐3 moiety, aquaporins, and calmodulin can cascade some brief pulses of rapid signalling events associated with changes in root physiology and metabolism. Stress and defence‐related proteins like HSPs, jacalins or non‐expressor of pathogenesis‐related gene 1 are also important in the process of root response to abiotic stress. Although updated proteomic approaches have led to the identification of oxiPTMs in various classes of root proteins, it is necessary to perform functional validation and regulation of the activity of each potential target protein. Such investigations shall provide a more detailed picture of the role of these oxiPTMs and their interconnections associated with root development and signalling.

Figure 4.

Root development and signalling in the differentiated zone (DZ), elongation zone (EZ), and meristematic zone (MZ) associated with thiol‐based oxiPTMs (PER, persulfidation; SNO, S‐nitrosation; and SU, S‐sulfenylation) of root proteins mediated by NO, H₂S and H₂O₂ under normal or challenging environments (salinity, nitrate stress or H₂O₂‐induced oxidative stress). Abp1, endoplasmic reticulum auxin binding protein 1; Axr, auxin resistant; Dlf1, dwarf in light 1; Lsf2, Like sex four 2; Pme, pectin methyl esterase; RbohD, respiratory burst oxidase homolog protein D; RC, root cap; tsb1, tryptophan synthase beta‐subunit; Vnd7, Vascular‐related nac‐domain7; Wes1, indole‐3‐acetic acid amido synthetase; Xet, Xyloglucan‐endotrans‐glucosylase/hydrolase 2‐like. [Color figure can be viewed at wileyonlinelibrary.com]

NO and H₂S signalling in plants shows interdependency, where endogenous H₂S levels are reported to be decisive for cascading NO‐mediated effects (Da‐Silva et al., 2018; Iqbal et al., 2021). Advancements in the methods for the detection of S‐nitrosated and persulfidated proteins have led to the possible understanding of interrelations among these two oxiPTMs. A total of 639 proteins in Arabidopsis have been reported to be targets of both S‐nitrosation and persulfidation (Hu et al., 2015). Although similar enzymes might have target sites for both oxiPTMS, certain proteins like RBOHD exhibit opposite regulatory effects mediated by S‐nitrosation and persulfidation, respectively (Shen et al., 2020; Yun et al., 2011). Thus, it is probable that these two oxiPTMs mediated by NO and H₂S are likely to exhibit synergistic or competitive regulatory effects on various proteins in a plant cell. Recent investigations have attracted attention to the physiological effects of S‐nitrosothiol thionitrous acid (HSNO), which emerges from an interaction between NO and H₂S (Marcolongo et al., 2019, Marozkina & Gaston, 2020). Thus, it is essential to undertake future investigations on the role of HSNO, which might trigger any potential oxiPTMs with regulatory roles on proteins.

4. NO, H₂S, AND H₂O₂ REGULATE ROOT SIGNALLING DURING ABIOTIC STRESS

Apart from organelle metabolism, a substantial amount of H₂O₂ is generated in the root apoplasts by the RBOH. H₂O₂ goes into the cytoplasm and triggers several secondary signalling pathways associated with an altered redox state of the cell (Noctor et al., 2018). Likewise, NO and H₂S due to their small size and lipophilic properties can also go through the cell membranes and participate in the mechanism of the response to multiple abiotic stresses such as salinity, drought, heavy metal/metalloid stress, or anoxia (D. Piacentini, Corpas, et al., 2020; Shivaraj et al., 2019; Zhu et al., 2018). Similar to the aquaporin‐facilitated movement of H₂O₂, NO and H₂S are also involved in the transport and regulation of the aquaporin activity (Bestetti et al., 2018; Kumari & Bhatla, 2021; Liu et al., 2007; Rodrigues et al., 2017). However, unlike animal systems, less information is available on the roles of NO and H₂S in mediating the expression and activity of aquaporins in plant roots.

APX isozymes exhibit spatial distribution at the subcellular level in plant tissues (cytosol, chloroplasts, mitochondria, and peroxisomes), where NO and H₂S‐mediated PTMs mediate positive and negative effects of the enzyme activity (Aroca et al., 2015, Begara‐Morales et al., 2013a; González‐Gordo et al., 2022; Keyster et al., 2011). Interesting findings through pharmacological and genetic analyses reveal that ROS‐MPK6‐NO signalling regulates NaHS sensitivity on root growth. Since a higher concentration of NaHS is inhibitory to root growth, a flow in ROS and NO levels are accompanied by higher activity of MPK6 during NaHS‐induced inhibition of primary root growth (Zhang et al., 2017). NaHS‐induced effect on root growth is mediated by RBOH activity and ROS outburst which trigger NO formation. Thus, MPK6 activity and ROS accumulation are the upstream events to NO signalling as evident during NaHS‐mediated regulation of primary root growth.

H₂O₂ levels generated from plasma membrane‐associated RBOH activity mediate NO signalling during adventitious rooting in arsenic‐stressed rice seedlings subjected to NO donor and scavenger (Kushwaha et al., 2019). This finding was accompanied by the lower APX activity, modulating H₂O₂ homoeostasis in rice roots. NO and H₂O₂ are known to be generated under similar situations of stressful environments where the formation of both biomolecules share similar kinetics (Niu & Liao, 2016). Oxalate oxidase analysis in maize plants performed through EM‐imaging and enzyme staining demonstrated that apoplastic H₂O₂ production regulates cell division and primary root elongation in the apical root growth zone of maize plants raised under normal and water stress conditions (Voothuluru and Sharp, 2013; Voothuluru et al., 2020).

H₂O₂ initiates NR‐mediated NO biosynthesis during lateral root formation in Arabidopsis involves the activity of MAPK (Wang et al., 2010). Mutant seedlings, namely mpk6‐2 and mpk6‐3, exhibited increased lateral root proliferation in the presence of exogenous NO and/or H₂O_2. MPK6 and NIA, therefore, appear to be redox‐regulated components that function as molecular switches for NO‐induced phenotypic plasticity of roots in Arabidopsis in the presence of H₂O₂ (Wang et al., 2010).

H₂O₂ accumulation is a downstream signal during its crosstalk with NO during the biochemical analysis of arsenic stress tolerance in soybean seedling roots (Singh et al. 2020). Endogenous H₂O₂ formation is crucial for NO‐induced regulation of the ascorbate‐glutathione cycle during arsenic toxicity. On the other hand, a negative correlation has been observed between NO and H₂O₂ levels during oxytetracycline antibiotic‐induced suppression of root growth in tomato seedlings where the authors used fluorescence methods followed by gene expression analysis of root meristem growth (Yu et al., 2017). H₂O₂ accumulation in the root apical meristem was attributed to induced root growth, which was, however, disturbed by oxytetracycline‐induced NO accumulation. Thus, NO‐induced disruption in H₂O₂ accumulation was also accompanied by cell cycle arrest and suppressed root growth (Yu et al., 2017).

By applying donors and specific inhibitors of H₂S and H₂O₂ synthesis, it has been shown that during mung bean seed germination and radicle formation, the interaction between H₂S and H₂O₂ promoted the seed germination by mobilizing reserve protein, where H₂O₂ functioned as a downstream signal molecule of H₂S (Li & He, 2015). Treatment with NaHS followed by monitoring the gene and protein expression of H⁺‐ATPase and plasma membrane Na⁺/H⁺ antiporter revealed that in the presence of salinity stress, the H₂S effect on ion homoeostasis (H⁺‐ATPase activity and Na⁺/H⁺ antiport expression) is mediated by the downstream H₂O₂ signalling in (Y.J. Li et al., 2014). Therefore, H₂O₂ is an important signalling molecule in the H₂S‐induced salinity tolerance pathway of Arabidopsis roots, where elevated expression of H⁺‐ATPase and Na⁺/H⁺ antiporters during H₂S supplementation occurs in an H₂O₂‐dependent manner. Although H₂O₂ signalling is a downstream component in H₂S‐mediated pathways (Z.G. Li & He, 2015, Y.J. Li et al., 2014, F. Liu et al., 2020), on the other hand, spermidine‐induced H₂O₂ signalling functions upstream to H₂S and NO‐mediated regulation of gene expression in dehydration‐stressed white clover plants (Z. Li et al., 2019).

Cys metabolism and H₂S signalling have sequential effects on H₂O₂ homoeostasis and sensitivity to abiotic stress in roots (M.C. López‐Martín, Becana, et al., 2008; M.C. López‐Martín, Romero, et al., 2008). H₂S‐mediated lateral root emergence and elongation in tomato seedlings is regulated by the increased RBOH1 expression (Mei et al., 2017). This is also accompanied by the regulation of cell cycle genes SlCYCA2;1, SlCYCA3;1, and SlCDKA1. Thus, in the H₂S‐facilitated developmental pathway of lateral root formation in tomato, H₂O₂ accumulation by the activity of RBOH1 regulates root primordia development and lateral root elongation. Using the H₂S‐specific fluorescent probe WSP‐1 (Washington State Probe‐1), it was found high H₂S content in the primordia region of tomato roots treated with NaHS (J. Li et al., 2014). Furthermore, the response mediated by H₂S is a downstream signalling event to IAA and/or NO‐mediated response. Although H₂O₂ is essential for H₂S‐mediated root growth regulation, hypoxia‐stressed pea plants show improved root tip growth during exogenous NaHS treatment which further reduces H₂O₂ levels (Cheng et al., 2013). Similarly, during waterlogged conditions in peach plants, enhanced H₂O₂ accumulation was attributed to root tip death, which was, however, alleviated by exogenous NaHS (Xiao et al., 2020). Table 4 summarizes several signalling events associated with NO, H₂S, and H₂O₂ crosstalk in plant roots under abiotic stress where H₂O₂ accumulation is a crucial signalling step in NO and H₂S mediated response during abiotic stress sensing in plant roots.

Table 4.

Examples of the implication of NO and H₂S donors in plants systems and their interactions

Plant systems and study conducted	Implied donors (NO and H2S)	Interactions	References
Citrus aurantium L. proteomic analysis and PTMs in salinity stress	SNP (100 µM)	Additive effects of NO and H2O2	Tanou et al. (2009)
Zea mays alleviation of Cr(VI) stress	SNP or NaHS (500 µM)	Alternatives with similar effects	Kharbech et al. (2017)
Zea mays alleviation of methylglyoxal toxicity under Cr(VI) stress	SNP or NaHS (500 µM)	Alternatives with similar effects	Kharbech et al. (2020)
Zea mays alleviation of thermos stress	SNP (150 µM), NaHS (500 µM) GYY4137 (500 µM) H2O2 (100 µM)	Similar effects of NO and H2S on H2O2‐mediated response	Li et al. (2015)
Oryza sativa lateral root development	H2O2 (5 µM)	Growth promotive	Chen et al. (2021)
Oryza sativa	NaHS (2 µM)	Reduces H2O2 content	Zhu et al. (2018)
Alleviation of Al stress
Hordeum vulgare reduction of Cd stress	NaHS (200 µM)	Reduces H2O2 content	Fu et al. (2019)
Glycine max alleviation of Al stress	NaHS (50 µM)	H2S functions downstream of NO	Wang et al. (2019)
Triticum aestivum salinity stress	NaHS (50 µM)	Suppress ROS content	Deng et al. (2016)
Helianthus annuus‐ROS signalling	SNP, DETA, SNAP, CAY (500 µM)	NO‐induced ROS generation	Singh and Bhatla (2017)

Abbreviation: ROS, reactive oxygen species.

Although H₂O₂ accumulation in smaller amounts might be beneficial as a signalling molecule, uncontrolled excess of ROS during oxidative stress results in inhibitory effects to root elongation. However, ROS overproduction has also physiological functions. For example in the Arabidopsis root apical meristem, salicylic acid promotes quiescent centre cell division through ROS accumulation (Wang et al., 2021). Thus, NO and H₂S might function in activating or reducing H₂O₂ levels. Most investigations have been observed to imply exogenous pharmacological treatments of NO or H₂S donors which have a regulatory effect on H₂O₂ metabolism. However, the effect of these biomolecules in exogenous treatments may not always mimic the in planta interactions (Shi et al., 2007; Zhang et al., 2017).

5. RHIZOSPHERIC INTERPLAY OF NO, H₂S, AND H₂O₂: INSIGHTS TO ROOT‐MICROBE INTERACTION

The rhizospheric NO is expected to modulate root growth and plant metabolism in soils rich in organic matter. Root‐generated NO and soil organic matter are important regulators of rhizospheric microbe metabolism (Gupta et al., 2014; Magdoff & Weil, 2004). Thus, there are possible links among soil organic matter, NO generation, and rhizosphere microflora which affect root growth and development. The application of H₂O₂ to irrigation solutions has been beneficial in improving the oxygenation status of clayey soils. Poor drainage in clayey soils is especially unsuitable for agriculture, wherein metabolism in roots is affected due to anoxic conditions. The use of 600‐800 ppm H₂O₂ in irrigation solution had beneficial effects on the growth and metabolism of pepper (Piper nigrum) plants (Ben‐Noah & Friedman, 2016). The class I haemoglobin proteins are essential carriers of NO which diffuses into the cytosol from the organelles. Thus, NO produced in root cells not only alters H₂O₂ homoeostasis but also transduced response by long‐distance signalling (Farnese et al., 2016). The use of several NO donors and scavengers resulted in differential modulation of ROS and RNS in sunflower seeding roots and cotyledons. Consequently, the roots that are in direct contact with the solutions are more sensitive to the NO donor/scavengers, while the differential effects are not significantly transmitted to cotyledons (Singh & Bhatla, 2017). Table 4 provides some examples of NO and H₂S donors used for various plant systems and their interactions with H₂O₂ signalling. Thus, response to H₂O₂, NO, or H₂S donor applied to root or in foliar spray might vary in terms of their response during long‐distance signalling. The application of these priming molecules in irrigation medium to roots might be promising in crop management and resilience to climate change.

H₂O₂ is a signalling molecule associated with legume symbiosis, arbuscular mycorrhizal colonization, and endophyte association in plant roots (Andrio et al., 2012; Arthikala et al., 2014; Espinosa et al., 2014; Kiirika et al., 2012; Mesa et al., 2009; Montiel and Quinto, 2019; Puppo et al., 2013). Although several reports decipher the NO function in the establishment of the symbiotic association in roots (Damiani et al., 2016; Hu et al., 2021; Kang et al., 2022; Martínez‐Medina et al., 2019) there is still insufficient information to conclude the associative role of NO and H₂S in regulating H₂O₂ signalling during root‐microbe interactions.

Arbuscular mycorrhizal exerts regulatory effects on ROS metabolism in the colonized root segment of various plants (Dhawi et al., 2017; González‐Guerrero et al., 2010; Gu et al., 2019; Wu et al., 2014). In certain cases, arbuscular mycorrhizal reduces H₂O₂ content by imposing a stronger antioxidant system which facilitates the mutualistic association of the fungi with roots. The generated H₂O₂ is counteracted by antioxidant enzymes which regulate the intensity of oxidative burst in the cells (Garg & Chandel, 2015; Hajiboland et al., 2019; Saroy & Garg, 2021; Q.S. Wu et al., 2006, 2007; Z. Wu et al., 2014). Interestingly, the signalling role of H₂O₂ is associated with the intercellular colonization of Glomus intraradices hyphae in the root cortical cells of Medicago truncatula (Salzer et al., 1999). Strong H₂O₂ accumulation was observed in the arbuscular containing cortical cells and hyphal tips penetrating the root cells. Thus, H₂O₂ accumulation is associated with spatial‐temporal colonization of fungal cells in roots and no significant H₂O₂ accumulation was observed in non‐colonizing appressoria or vesicles (Salzer et al., 1999). This was accompanied by a higher peroxidase activity in the colonized segments of roots. Physiological and biochemical data indicate that higher H₂O₂ accumulation is triggered in response to drought stress in roots and nodules of peanuts associated with Bradyrhizobium SEMIA6144 symbiosis (Furlan et al., 2012; Jeon et al., 2011).

Using overexpressing transgenic lines and monitoring the nodulation‐associated transcript levels, it was found that the RBOHs exhibit dual function as both positive and negative regulators of symbiotic and mycorrhizal association (Arthikala et al., 2014). Analysis of Phaseoulus vulgaris overexpression lines for PvRbohB revealed that a higher amount of O₂ ^•− generation was associated with improved nodule biomass, early nodulin expression, and N‐fixation, but significantly impaired mycorrhizal colonization (Arthikala et al., 2014). The intensity of H₂O₂ accumulation in different zones of pea roots is a determinant of pathogenesis or mutualism response brought about by different strains of Rhizobium leguminosarum bv. viceae (Kuzakova et al., 2019). In gymnosperms like Pinus sylvestris, inoculation with necrotrophic fungi (Fusarium oxysporum; Rhizoctonia solani) and mycorrhizal fungi (Hebeloma crustuliniforme and Laccaria bicolor) revealed spatial differences in H₂O₂ accumulation in the elongation and meristematic regions of roots, wherein, infective fungi triggered the higher accumulation of H₂O₂ as a signalling event within 48 hpi (Mucha et al., 2019). Thus, H₂O₂‐associated oxidative burst is a signalling response in root‐microbe recognition both during mutualistic or infective responses.

NO–H₂O₂ association mediates the infection establishment and nodule formation in roots, wherein, S‐nitrosation and S‐sulfenylation of different proteins in the early and later stages of nodule initiation reveal redox‐regulatory mechanisms (Melo et al., 2011, Oger et al., 2012, Puppo et al., 2013). Several RBOH and NR mutants have been used for the functional validation of the H₂O₂ and NO implication in the course of nodule initiation and development. Medicago truncatula roots treated with the RBOH inhibitor diphenyleneiodonium and NO scavenger cPTIO revealed that 316 differentially expressed genes were common for both treatments and were associated with cell wall formation, plant defence, and secondary metabolism (Puppo et al., 2013). This finding suggests potential crosstalk mechanisms between NO and H₂O₂ during legume‐bacterial symbiosis. Using a genetic approach, the authors conclude that phytoglobin 1‐mediated NO regulation is prevalent during arbuscular mycorrhizal symbiosis between tomato–Rhizophagus irregularis (Martínez‐Medina et al., 2019). A specific pattern of NO accumulation was observed in the tomato roots undergoing arbuscular mycorrhizal symbiosis which was also accompanied by an upregulation of phytoglobin 1 (Martínez‐Medina at al., 2019). The RBOH genes are needed for cascading signals for nodule formation and infection thread ingression in root hairs (Damiani et al., 2016; Montiel et al., 2012). Furthermore, ROS production and H₂O₂ burst have been observed to be a downstream response to infection and recognition by the host root which facilitates the advancement of the infection thread, but higher levels of ROS at the later stages might be inhibitory to nodulation (Cárdenas et al., 2008; Montiel et al., 2012; Santos et al., 2001; Tóth & Stacey, 2015). The increased H₂O₂ levels during nodule formation can regulate the transcription of enzymes involved in NO homoeostasis (Andrio et al., 2012). NO and H₂O₂‐mediated chemical interactions are beneficial during plant‐endophyte interactions. Thus, microbe‐generated ethylene triggers H₂O₂ accumulation in plant root hairs which in turn cascades NO production by the intracellular bacteria (Chang et al., 2021). In this context, ethylene seems to be an upstream molecule in inducing H₂O₂ and NO biosynthesis in the root‐bacterial interface.

H₂S participates in regulating the nitrogen‐fixing ability of soybean root nodules colonized by Sinorhizobium fredii. Increased H₂S content upregulates nitrogenase activity in the N‐fixing zones of root nodules (N.N. Zhang et al., 2020; Zou et al., 2020). Furthermore, H₂S modulates the H₂O₂ content in root nodules and exerts positive effects in regulating N‐fixation, nodule number, and senescence‐associated genes in N‐deficient soybean plants in association with rhizobia (Alesandrini et al., 2003; N.N. Zhang et al., 2020). Exogenous NaHS increased nodulation and nitrogenase activity, furthermore, H₂S positively upregulated the expression of nodulation‐associated genes nodA, nodB, and nodC in S. fredii (Zou et al., 2019). H₂S is a positive regulator of the symbiosis‐associated signalling events during the soybean‐rhizobium association. H₂S was associated with the infection threads in root hairs of infected roots. Furthermore, by monitoring the fluorescent localization of H₂S it was deciphered that H₂S downregulated NO and ROS accumulation while increasing H₂S content in roots (Fukudome et al., 2020). Figure 5 depicts the signalling routes of NO, H₂S, and H₂O₂ associated with root nodulation, mycorrhizal association, and endophytic associations.

Figure 5.

NO, H₂S, and H₂O₂ exhibit mutual interactions among themselves which promote the various instances of root‐microbe associations such as legume‐rhizobium symbiosis, endomycorrhizal association, or endophytic growth in plant roots. The activities of antioxidant enzymes like superoxide dismutase (SOD), peroxidase (POD), or catalase (CAT) are jointly regulated by the activity of NO and H₂S. Thus redox homoeostasis is important for establishing the initial phases of infection/mutualistic association among the root and microbes. NO and H₂S‐mediated PTMs of nodulation‐specific proteins assist in the process of legume‐rhizobium interaction. NADPH oxidase and nitrate reductase activity are modulated by both NO and H₂S which facilitate the expression of nodulation genes, the establishment of symbiosis, and nodule maturation. Endomycorhizal growth in root cells is facilitated by a higher accumulation of H₂S and H₂O₂. Endophytic growth in roots triggers ethylene biosynthesis in the root hair‐microbe interfaces which also promotes H₂O₂ accumulation. NO and H₂S‐producing soil microbes are associated with different pathways of anaerobic metabolism which contributes to a considerable amount of NO and H₂S emissions in marshy wetland soils rich in organic content. This soil‐based source of exogenous gasotransmitters participates in root‐microbe signalling under normal and physiologically challenging environments. [Color figure can be viewed at wileyonlinelibrary.com]

ROS outburst during host‐microbe recognition facilitates the process of its establishment in the roots. Nevertheless, NO and H₂S‐dependent signalling routes and their association with ROS levels have gained importance in the context of the root‐microbe association. NO‐H₂S crosstalk facilitates a surge in nitrogenase activity in the nodulation stages. The upregulation of nodulation genes NODA, NODB, and NODC is mediated by the interaction of NO and H₂O₂. The changes in H₂O₂ levels are connected with the expression of genes related to NO homoeostasis in the root nodules. H₂S signalling participates in the process of infection thread formation between the bacteria and legume roots. Cell wall formation, plant defence regulation, and biosynthesis of secondary metabolites are the prerequisites for the establishment of the mycorrhizal association, wherein H₂S plays a role in modulating antioxidant enzymes and NR activity. H₂S metabolism is crucial for modulating ROS content which in turn regulates microbe‐mediated ethylene formation at the root‐soil interface. Thus, rhizospheric associations accompany transient ROS outburst which is sequentially regulated by NO and H₂S‐mediated modulation of root growth, microbial recognition, modulation of metabolic proteins, and phytohormonal crosstalk.

6. FUTURE PROSPECTS

Identifying novel metabolic signatures associated with NO, H₂S, and ROS interactions associated with root development, phenotypic plasticity, and stress tolerance is a challenge for future research. Although soluble guanylate cyclase has been reported to be a potential NO receptor in the animal system (Martin et al., 2005) the functional homolog of natriuretic peptides in plants has been proposed to be a functional form of guanylate cyclase which involves cGMP‐dependent signalling (Hsiao and Yamada, 2020; Turek & Gehring, 2016). However, research in plant systems is still waiting for the functional characterization of a specific NO receptor. Likewise, plant H₂S signalling includes pharmacological studies associated with exogenous donors that regulate metabolism, hormone signalling, and gene expressions. Unlike animal systems, no reports have been found on the molecular mechanisms of receptor‐mediated H₂S signalling in plant cells. Although H₂O₂ sensor HPCA1 has been characterized to be an LRR receptor kinase in Arabidopsis (Wu, Chi, et al., 2020), it is important to investigate the role of NO and H₂S signalling in the regulation of the HPCA1 function.

Future studies must focus on the identification of the oxiPTMs mediated by NO, H₂S, and H₂O₂ in specific developmental stages of primary root (meristem differentiation), lateral root primordia initiation, extension, and adventitious rooting. While several fluorescent probes have been used to study the cellular NO content and distribution, few works have monitored H₂S in plant tissues. It is necessary to know how NO, H₂S, and H₂O₂ interactions modulate the activity of some target proteins involved in stress response, cell wall extension, and root phenotype plasticity under challenging environments. Several questions remain unanswered on the priority or abundance of the oxiPTMS in roots mediated by endogenous NO, H₂S, and H₂O₂ under different environmental conditions. Research should focus on deciphering the molecular pathways of the interaction of the Ca²⁺‐CaM complex at the crossroads of NO, H₂O₂, and H₂S signalling in roots. Since aquaporins are involved in crucial signalling events in root cells under abiotic stress, research should evaluate how NO, H₂S, and H₂O₂ could regulate the expression and activity of aquaporins in root cells. Future works on NO‐H₂S interaction must explore the potential function of polysulfide in intracellular and long‐distance signalling in plant tissues.

Since nitrate deficiency influences ROS levels associated with the proliferation of root apical meristem, it would be necessary to explore strigolactone‐NO and ROS interactions in nitrate‐deficient roots (Sun et al., 2016). New studies on the interplay of strigolactones and H₂S are likely to reveal their role in H₂O₂‐mediated root development and signalling in plants (Bharti et al., 2015). Considering that ethylene has been recently known to be involved in both regulations of H₂O₂, and karrikin signalling (Carbonnel et al., 2020), it would be fruitful to consider the role of ethylene‐karrikin interaction in regulating ROS, NO, and H₂S levels in roots. The dynamics of NO and H₂S generation in various organelles of root cells require also further attention. In this context, it is also essential to get a much clear picture of the coordination of ROS levels among apoplast, cytosol, and organelles in the cells. Genetically encoded ROS, NO, and H₂S sensors in several regions of the root might help to map their spatial distribution. Despite the advance obtained in the last decade on NO, H₂S, and H₂O₂ signalling in root physiology, several challenges exist in the measurement of transient NO and H₂S flux from the rhizosphere in different soil and climatic conditions. Furthermore, the measure of the dynamic fluctuations in NO and H₂S levels in plant root tissues also involves suitable expertise. No investigations to date substantiate the role of soil‐based NO and H₂S emissions in the regulation of root phenotype in marshy wetlands or nitrate/sulphate‐rich soils. Detailed future studies in specific plants like Arabidopsis or crops like rice or sunflower might enable us to understand the interaction of these signalling molecules under submerged (anoxia) and other stress conditions.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Mukherjee, S. & Corpas, F.J. (2023) H₂O₂, NO, and H₂S networks during root development and signalling under physiological and challenging environments: beneficial or toxic? Plant, Cell & Environment, 46, 688–717. 10.1111/pce.14531

DATA AVAILABILITY STATEMENT

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.