The role of H₂S and its derivatives in plant responses to low O₂

Abstract

Hydrogen sulfide (H₂S) is a gasotransmitter molecule that regulates essential plant biological processes. In the context of climate change and increasing flooding events, its role in plant responses to oxygen (O₂) deficiency has gained significant attention. Accumulating evidence indicates that H₂S exerts beneficial effects under hypoxia by modulating antioxidant defenses, mitochondrial function, and hormonal signaling, mainly through its ability to refine protein activity via the post-translational modification (PTM) of persulfidation. Here, we discuss how this PTM is linked to the gasotransmitter molecule nitric oxide (NO) and reactive oxygen species (ROS), and how they can act synergistically or antagonistically, influencing the cell redox state and survival. Additionally, we examine how H₂S turnover is intrinsically linked to O₂ availability, acting as a conserved mediator of low O₂ responses in different domains of life. By integrating biochemical insights with molecular signaling, we underscore the potential of H₂S in enhancing crop resilience to low O₂-related stresses, such as submergence and waterlogging.

Through a comprehensive and critical evaluation of the available literature, we compare and discuss contrasting data to better define the molecular mechanisms through which H₂S may influence low-oxygen signaling pathways.

Introduction

Climate change is increasing the frequency of environmental disasters, which have a devastating effect on global agriculture. A recent report from the United Nations Food and Agriculture Organization states that from 1991 to 2023, disasters caused US$3.6 trillion damage, with flooding being the most economically destructive hazard (US$1.5 trillion losses over the same period) (FAO, 2025). Strategies to improve crop resilience to flooding are necessary to mitigate crop losses, which can be facilitated by an understanding of plant molecular responses to submergence.

A significant stressor upon plant submergence is the reduction in O₂ availability, which limits aerobic respiration and efficient ATP production. The molecular response to reduced O₂ availability, which directs a switch to anaerobic metabolism, has been well characterized and is described in detail in many recent reviews (Voesenek and Bailey-Serres, 2015; Loreti and Perata, 2023; Renziehausen et al., 2025). However, flooding is a compound stress that includes the accumulation of other gaseous signals, as well as signals initiated by other factors such as darkness and energy availability (Renziehausen et al., 2024). Major gaseous compounds involved in cell signaling during flooding are nitric oxide (NO) and the hormone ethylene (Sasidharan et al., 2018; Hartman et al., 2019). Lately, a role is emerging for a third molecule, hydrogen sulfide (H₂S).

H₂S (also termed sulfane) is a soluble, volatile and cell-permeable gas which is toxic at high concentrations. It is only in the last 30 years that it has been revealed to have a role in cell physiology (Wang et al., 2022). Although H₂S itself does not accumulate to high concentrations, H₂S-derived signals can affect a range of physiological processes which, in plants, have been reported to act as important signals in development, growth, and stress responses, described in detail in several reviews (Paul and Snyder, 2012; Aroca et al., 2015; Gotor et al., 2019; Moseler et al., 2024). Some cornerstone proteomic studies have demonstrated that protein post-translational modification (PTM) is the main mechanism for H₂S-mediated signaling in plants (Aroca et al., 2017, 2020; Romero et al., 2026). Persulfidation can affect the localization, conformation, dimerization, or activity of proteins, according to its particular targets, thereby exerting a range of profound effects on cell regulation (Zhou et al., 2025).

The close relationship between O₂ availability and H₂S oxidation suggests that H₂S-derived signals may be indicators of hypoxia and oxidative stress. However, the dynamic reactivity of H₂S-derived species, including with prevalent intracellular thiols such as Cys residues and glutathione (GSH), makes these modifications challenging to study. The modes of action of H₂S-based signaling are therefore not yet fully understood. In this review, we summarize current understanding of the role of H₂S in plant physiology, with a focus on its involvement in low O₂ signaling. We discuss the range of H₂S-dependent PTMs and their dynamics as well as how these are affected by changes in O₂ availability and redox status. Finally, we propose that H₂S-derived signals may play a more prevalent role in dynamic plant stress responses than previously thought. However, careful analysis and accurate characterization of H₂S-derived persulfidation reactions are essential to reconcile the complex chemical and biological features of this fascinating molecule.

Hydrogen sulfide as a gasotransmitter

From toxic molecule to gaseous signal

Gasotransmitters are small, endogenously produced gaseous molecules able to serve as signaling messengers (the neologism first appeared in an article referring precisely to H₂S, as a contraction of ‘gaseous transmitters’; Wang, 2002). The concept has emerged from a series of discoveries that revolutionized cell biology in the late 20th century. For decades, gases such as NO, carbon monoxide (CO), and H₂S have been considered environmental pollutants or metabolic byproducts rather than signaling molecules. The turning point came in the 1980s, when NO enzymatic synthesis by nitric oxide synthase (NOS) was demonstrated and its functions as a key regulator in human physiology were identified, as a mediator of the relaxation of vascular smooth muscle caused by endothelium-derived relaxing factor (EDRF) (Ignarro et al., 1987). These findings earned the authors the 1998 Nobel Prize in Physiology or Medicine and introduced the notion that small gaseous molecules can act as signaling messengers.

Following this conceptual breakthrough, researchers began exploring whether other gas molecules could serve a role in signaling. H₂S, long recognized for its characteristic odor and toxicity, became a candidate. It was reported in 1996 that brain tissues produce measurable H₂S levels and that this can modulate N-methyl-D-aspartate (NMDA) receptor activity, marking the first evidence of H₂S-derived signals as a neuromodulator (Abe and Kimura, 1996). Subsequent work has identified cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST) as enzymes responsible for H₂S biosynthesis in mammals (Hosoki et al., 1997). More recently, the H₂S-producing activity of the methanethiol oxidase (MTO) SELENBP1 has been discovered in humans (Pol et al., 2018). By the early 2000s, H₂S had thus been recognized as the third gasotransmitter in humans, following NO and CO (Wang, 2012). The chemical and physical properties of these molecules allow them to diffuse through membranes and act independently of vesicular trafficking or receptor binding (Mustafa et al., 2009b). They can accumulate locally, integrating environmental and metabolic stimuli into cellular signaling (Mustafa et al., 2009b). Moreover, another distinctive feature of gasotransmitters is their high instability and chemical reactivity (Wang, 2014).

In plants too, NO, CO, and H₂S are established gasotransmitters, involved in stress responses and development (Kolupaev et al., 2022). The identification of methane and molecular hydrogen (H₂) as additional gasotransmitters, both found to be connected to stress responses, is presently hindered by the lack of an identified mechanism of biosynthesis and action in plants (Yao et al., 2019; L. Li et al., 2020). Ethylene is another gas ubiquitously generated by plants that covers paramount functions in signaling. Despite this, ethylene, being a phytohormone, rather eludes the definition that describes gasotransmitters as reactive molecules that function independently of receptors (Wang, 2014). The discovery of H₂S as a plant gasotransmitter, only preceded by the identification of NO, dates back to the observation of H₂S emissions from leaves of several plant species (Wilson et al., 1978). As in animal systems, H₂S in plants meets the defining criteria of being a small membrane-permeable gas, generated by endogenous and regulated enzymatic pathways, having high reactivity, and interacting with signaling pathways (directly or with the mediation of hormones) (Wang, 2002). Overwhelming evidence has built up over time that shows H₂S participation in numerous signaling pathways (Aroca et al., 2020; Xuan et al., 2020; Wang et al., 2022) during stress and growth responses that are mediated, among other signals, by hormones (Fang et al., 2014; Aroca et al., 2021; Yuan et al., 2025), Ca²⁺ (Fang et al., 2017), NO (Corpas et al., 2019; Mukherjee and Corpas, 2020), and reactive oxygen species (ROS) (Shen et al., 2020; Xiao et al., 2020).

Biosynthesis and signaling role of hydrogen sulfide in plants

H₂S in plants is mainly produced by cysteine desulfhydrase enzymes (CDes) (Rennenberg et al., 1987), including L-cysteine desulfhydrase (LCD) and the bifunctional cystathionine γ-lyase/cysteine synthase [DES1, a member of the O-acetylserine thiol lyase (OASTL) family]. CDes decompose L-Cys into H₂S, pyruvate, and NH₄⁺ using pyridoxal 5'-phosphate (PLP) as cofactor. Among them, DES1 is a cytosolic enzyme (Álvarez et al., 2010), while the LCD family encompasses both cytosolic and nuclear isoforms (González-Gordo et al., 2020; Hu et al., 2020; Cao et al., 2022) (Fig. 1). Chloroplasts can also generate H₂S through sulfite reductase (SiR), during the reductive steps of sulfate assimilation (Fig. 1). At the alkaline pH typical of the stromatic environment under illumination, H₂S (pK_a=6.98) (Vignane and Filipovic, 2023) predominantly exists as soluble hydrosulfide (HS⁻) and is unable to cross the chloroplast envelope in the absence of dedicated transporters (Gotor et al., 2019). Hence, H₂S release from this organelle is believed to be negligible, but the molecule can still have a broad impact on the regulation of plastidial pathways (Aroca et al., 2015; Corpas et al., 2019). A third source of H₂S is represented by the mitochondrial β-cyanoalanine synthases (β-CASs), such as CYS-C1 in Arabidopsis, the prevalent isoform in this family (Watanabe et al., 2008). β-CASs, PLP-dependent enzymes that convert Cys and cyanide to H₂S and β-cyanoalanine, are required to maintain endogenous cyanide at low levels compatible with physiological processes (Machingura et al., 2016) (Fig. 1). However, the option that insufficient H₂S production could contribute to the alterations observed in β-CAS mutants during root hair formation or salt stress responses has not been addressed so far (García-Mata and Lamattina, 2010; Xu et al., 2023).

Fig. 1.

H₂S biosynthesis in plant cells. H₂S can be released in different cell compartments, with cytosolic desulfhydration being the prevalent pathway. SO₄²⁻, sulfate; SO₄³⁻, sulfite; NH₄⁺, ammonia; CN⁻, cyanide; SiR, sulfite reductase (AT5G04590); DES1, L-cysteine desulfhydrase 1 (AT5G28030); LCD, L-cysteine desulfhydrase (AT5G28020); OASTL, O-acetylserine(thiol)lyase isoform A1 (AT4G14880); β-CAS, β-cyanoalanine synthase C1 (AT3G61440). Dotted lines represent multistep processes. Created in BioRender. Flashman, E. (2026) https://BioRender.com/oy343jm.

Among the H₂S-producing routes, the DES1 pathway (the most recently identified pathway) is recognized as the prevalent source in plants, accounting for ∼30% H₂S production in Arabidopsis tissues (Álvarez et al., 2010; Aroca et al., 2017; Gotor et al., 2019). Knockout des1 mutants show early leaf senescence, enhanced defense responses against biotrophic and necrotrophic pathogens, and lower susceptibility to cadmium toxicity (Álvarez et al., 2010, 2012a). The isolation of des1 has paved the way for numerous studies that have demonstrated the involvement of H₂S in senescence, autophagy, stomatal closure, and immunity (Álvarez et al., 2010, 2012b; Scuffi et al., 2014; Laureano-Marín et al., 2016).

Part of the observed effects associated with the manipulation of H₂S levels have been related to Cys homeostasis and antioxidant responses. Low Cys desulfuration in des1 knockout plants leads to higher Cys content (Álvarez et al., 2010), while on the other hand Cys biosynthesis through OASTL action is known as a detoxification mechanism in the presence of excessive H₂S levels (Birke et al., 2015). The interplay between H₂S and Cys has consequences for redox homeostasis, through both GSH and the regulation of antioxidant enzyme activity. GSH is a Cys-containing tripeptide that acts as a primary antioxidant and key player in the redox balancing of cells (Noctor et al., 2011). The amelioration of plant survival promoted by both Cys and H₂S supplementation in the presence of heavy metal stresses can be partially explained by an improvement in redox homeostasis associated with higher Cys production, in addition to the synthesis of Cys-derived metal chelators, such as phytochelatins and metallothioneins (Fang et al., 2016, 2017; Jia et al., 2016). Nonetheless, the role of H₂S appears to go well beyond its participation in a metabolic feedback loop with Cys. H₂S- and Cys-treated Arabidopsis seedlings showed limited transcriptional overlap and H₂S-specific responses associated with transcriptional regulation, PTMs, and hormone signal transduction (Fang et al., 2023). This suggests that the mode of action of H₂S entails Cys-independent mechanisms, with protein persulfidation considered as the predominant mechanism by which H₂S-mediated signaling takes place (Vignane and Filipovic, 2023).

Post-translational protein modifications associated with hydrogen sulfide

H₂S, along with its oxidized derivatives, belongs to a broader group known as reactive sulfur species (RSS), a collective class of sulfur-containing compounds, analogous to ROS and reactive nitrogen species (RNS) (Cortese-Krott et al., 2017). Over the past two decades, RSS have been established as regulated mediators of redox signaling and oxidative stress, positioning sulfur as a central component of redox biology (Giles et al., 2017). The capacity of sulfur to span oxidation states from −2 (thiols, H₂S) to +6 (sulfate) (Fig. 2A) confers functional versatility, permitting sulfur-containing molecules to mediate diverse redox processes (Mishanina et al., 2015; Smith and Pluth, 2023). In cells, sulfur in oxidation state −2 functions primarily as a reductant and nucleophile. At −1 and 0, it forms disulfides (-SS), polysulfides (-SS_n-R), persulfides (-SSH), sulfenic acids (-SOH), and S-nitrosothiols (-SNO). These redox-dependent modifications, promoted in the presence of ROS, RNS, and/or RSS, play active roles in redox signaling. At higher oxidation states, sulfur occurs as sulfinic acids (-SO₂H), sulfonic acids (-SO₃H), and sulfate (SO₄²⁻), which are generally regarded as terminal oxidation products, with the notable exception of target-specific sulfinic acid reduction mediated by sulfiredoxin (Biteau et al., 2003; Mishanina et al., 2015; Akter et al., 2018; Filipovic et al., 2018).

Fig. 2.

Oxidation states of biologically relevant sulfur species and their functional roles. (A) Sulfur spans oxidation states from −2 to +6, enabling participation in reductive chemistry, reversible redox signaling via reactive sulfur species (RSS), and terminal oxidative products. Among the terminal oxidation states of sulfur, sulfinic acids represent an exception to irreversibility, as Cys-sulfinic residues can be reduced through target-specific sulfiredoxins. (B) Persulfidated Cys is usually formed after H₂S reaction with Cys sulfenic acid or S-nitrosocysteine residues in proteins. R-SH, thiol group; H₂S, hydrogen sulfide; HS⁻, hydrosulfide anion; R-SS-R, disulfide bond; R-SSH, persulfide group; R-SOH, sulfenic acid; R-SNO, S-nitrosothiol; R-SS_n-R, polysulfide chain; R-SO₂-H, sulfinic acid; R-SO₃-H, sulfonic acid; SO₄²⁻, sulfate ion. Created in BioRender. Giuntoli, B. (2026) https://BioRender.com/ush26ff.

Within the cellular redox landscape, H₂S is the most reduced biologically relevant sulfur species. It is maintained at low intracellular concentrations (∼10–30 nM in mammalian systems), consistent with its role as a tightly regulated and spatially confined signaling molecule (Furne et al., 2008; Vitvitsky et al., 2012; Kabil et al., 2014). H₂S is a weak acid and, in aqueous solutions at physiological pH, is present mainly as hydrosulfide anion (HS⁻), while recent studies indicate that S²⁻ does not exist in measurable amounts in aqueous solutions (May et al., 2018). Under these conditions, HS⁻ can function either as a ligand or as a redox-active sulfur donor, depending on the reaction context.

O₂ availability is a key determinant of H₂S turnover. Under aerobic conditions, H₂S is rapidly consumed through mitochondrial oxidation pathways and thiol-dependent reactions that promote its conversion into persulfides and other more oxidized forms (Olson, 2012; Filipovic et al., 2018). In contrast, under anaerobic or hypoxic conditions, oxidative H₂S catabolism is suppressed, resulting in lower mitochondrial and O₂-dependent turnover, greater persistence of H₂S/HS⁻, and diminished formation of RSS. This is consistent with a model in which H₂S and RSS metabolism is inversely correlated with O₂ tension and has led to the proposal that H₂S-derived reactions can act as an indirect O₂-sensing mechanism, at least in animal systems (Olson, 2021). The cellular redox state further governs reaction selectivity, as oxidizing environments favour reaction between H₂S/HS⁻ and more oxidized thiol species, thereby shaping RSS formation and downstream redox signaling processes (Cuevasanta et al., 2015; Wedmann et al., 2016).

H₂S interacts with proteins through two mechanistically distinct chemical processes: (i) reversible coordination of HS⁻ with iron centers in heme-containing proteins; and (ii) covalent modification of Cys residues via persulfidation. At physiological pH, HS⁻ readily coordinates with Fe²⁺/Fe³⁺ centers in proteins, forming transient ferric sulfide complexes that are unstable under aerobic conditions, as shown for the human hemoglobin, myoglobin, neuroglobin, and cytochrome c proteins (Jensen and Fago, 2018; Vitvitsky et al., 2018). Here, these complexes undergo oxidation, converting sulfide (−2) to thiosulfate (with sulfur in mixed −1/+5 oxidation states) and to metal-bound polysulfides (sulfur in mixed −1/0 oxidation states). These interactions can thereby modulate local H₂S availability while also generating RSS that can participate in redox signaling and antioxidant defense (Vitvitsky et al., 2015; Ruetz et al., 2017).

In parallel, persulfidation involves the reversible conversion of protein cysteine thiols (-SH, −2) into persulfides (-SSH, −1), a post-translational modification that enhances sulfur nucleophilicity, lowers the apparent thiol pK_a, and can protect Cys residues from irreversible overoxidation to -SO₂H or -SO₃H. Through these properties, persulfidation functions as a dynamic redox switch in cellular signaling (Mustafa et al., 2009a; Wedmann et al., 2016; Filipovic et al., 2018). Primary representatives of persulfidation are per- and polysulfides (R-SS_nH), where the R group denotes protein cysteine side chains and n is ≥1. Persulfidation can also occur on GSH and other low-molecular-weight thiols, contributing to the broader cellular persulfide pool (Ida et al., 2014). Importantly, H₂S cannot react directly with other reduced thiols, as the direct reaction is both thermodynamically unfavorable and kinetically slow. From a mechanistic perspective, a need for oxidative priming is consistent with the low reactivity of H₂S toward reduced thiols. Instead, persulfidation typically proceeds via oxidized Cys intermediates, such as sulfenic acids, disulfides, or S-nitrosothiols (Fig. 2B), or through reactions with other reactive sulfur species and polysulfides. Chemical and cellular studies support this model and show that persulfidation can act as a protective modification. Sulfenylation (the conversion of cysteine thiols into their first oxidized state, -SOH) is a reversible modification that regulates protein folding and function (Beedle et al., 2016; Huang et al., 2019). The unstable -SOH groups can be rapidly oxidized to Cys-sulfinic or -sulfonic acids, the latter at least being an irreversible process that is associated with protein oxidative damage. By resolving ROS-mediated sulfenylation, persulfidation prevents irreversible Cys overoxidation and helps preserve protein function under stress conditions (Filipovic et al., 2018; Zivanovic et al., 2019; Andrés et al., 2025). The rate constant for persulfide formation from H₂S and sulfenic acids (-SOH) is 270 M⁻¹ s⁻¹, which is markedly higher than that for reactions with disulfides (0.6 M⁻¹ s⁻¹). This kinetic difference indicates that protein persulfidation probably proceeds primarily through the reaction of H₂S with sulfenic acid groups. As indicated above, persulfidation modifications are subsequently reversible and tightly regulated by thioredoxin-, glutaredoxin-, and GSH-dependent systems (Dóka et al., 2016; Moseler et al., 2024). Notably, this pathway is kinetically favored in vivo because protein cysteine thiols are far more abundant than H₂S, and these thiols readily react with ROS, particularly H₂O₂, at rates that are orders of magnitude faster than with sulfide. Consequently, ROS act as critical facilitators of H₂S-mediated redox signaling by generating the oxidized thiol intermediates required for persulfidation (Filipovic et al., 2018; Benchoam et al., 2019; Zivanovic et al., 2019; Liu et al., 2024). H₂S-derived signaling via protein modification is therefore complex and must be experimentally analyzed with care and attention to the redox conditions in both the biological context and methodology (Li and Lancaster, 2013).

Protein persulfidation in plant physiology

Persulfidation targets in plant signaling and stress responses

Proteomic analyses show that protein persulfidation is a widespread PTM, affecting hundreds of proteins in mammalian systems and thousands in plants. Identified targets span central metabolism, antioxidant defense, transcriptional regulation, and signaling pathways, underscoring the broad regulatory scope of persulfidation in plant physiology (García-Mata and Lamattina, 2010; Aroca et al., 2017; Li et al., 2024). A major group of targets includes metabolic enzymes that coordinate energy and redox balance. For example, cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPDHs; GapC1 and GapC2) undergo inhibitory S-nitrosylation and S-glutathionylation at Cys150, whereas H₂S-induced persulfidation enhances GAPDH activity. Specifically, persulfidation at Cys160 promotes both catalytic activation and cytosol-to-nucleus translocation, linking metabolism to nuclear signaling (Zaffagnini et al., 2012; Aroca et al., 2015, 2017). Similarly, persulfidation of cytosolic glucose-6-phosphate dehydrogenase (G6PDH) in Arabidopsis (Cys159) and tomato (Cys155) modifies oligomeric structure, increases NADP⁺ affinity, and enhances activity, especially under salt stress (Wang et al., 2023). Persulfidation of succinate dehydrogenase (SDH, Complex II) stimulates mitochondrial respiration and ATP production, supporting guard cell energetics and stomatal regulation (Zhang et al., 2025). Additionally, persulfidation activates the H₂S-producing enzyme DES1 (Cys44, Cys205), increasing intracellular sulfide levels and contributing to downstream stress signaling (Shen et al., 2020).

Persulfidation also fine-tunes antioxidant systems that maintain cellular redox homeostasis. Cytosolic ascorbate peroxidase (APX1) in Arabidopsis is activated by persulfidation (Aroca et al., 2015; Wang et al., 2021). In tomato, specific modifications of APX1 (Cys234) and the class III peroxidase POD5 (Cys61) enhance their activities, whereas persulfidation of catalase (CAT1, Cys234) decreases activity. These differential effects suggest that this PTM dynamically redistributes antioxidant capacity to optimize ROS detoxification during stress (J. Li et al., 2020).

Autophagy-related proteins represent another important persulfidation module, linking cellular recycling to stress adaptation. During endoplasmic reticulum (ER) stress, persulfidation of ATG18a at Cys103 enhances lipid binding and inhibits autophagosome maturation, while abscisic acid (ABA)-induced modification of AtATG4a at Cys170 suppresses its protease activity and modulates autophagic flux (Laureano-Marín et al., 2020; Aroca et al., 2021). Persulfidation additionally regulates diverse stress proteins: modification of the BOI-related E3 ubiquitin ligase BRG3 alters ubiquitination and transcription factor interactions, and persulfidation of the plasma membrane H⁺-ATPase PMA1 promotes ion homeostasis and salt tolerance (Ma et al., 2023; Sun et al., 2023).

A prominent hub of persulfidation-dependent regulation is ABA signaling. A well-characterized example is the regulation of ABA responses in guard cells, in which H₂S modulates stomatal movements through persulfidation of key components such as SnRK2.6, DES1, and RBOHD (Chen et al., 2020; Shen et al., 2020). H₂S enhances ROS production through persulfidation of the NADPH oxidase RBOHD at Cys825 and Cys890, promoting ABA-induced stomatal closure (Shen et al., 2020). Concurrently, persulfidation of the protein kinase SnRK2.6 at Cys131 and Cys137 increases kinase activity, improves phosphate transfer efficiency at Ser175, and strengthens interaction with the transcription factor ABF2, synergistically amplifying ABA and drought responses (Chen et al., 2020, 2021). At the transcriptional level, persulfidation of ABI4 at Cys250 enhances its stability and regulatory activity, linking ABA signaling with mitogen-activated protein kinase (MAPK) cascades and controlling seed germination. Additional ABA-associated transcription factors, including WRKY and basic helix–loop–helix (bHLH) proteins, undergo persulfidation that modulates DNA binding and protein interactions, contributing to stress adaptation and developmental regulation, such as delayed fruit ripening (Liu et al., 2019; Zhou et al., 2021; Sun et al., 2023).

Beyond ABA, persulfidation shapes wider stress signaling pathways. Persulfidation of MPK4, probably occurring on multiple Cys residues, enhances kinase activity and supports cold stress tolerance (Du et al., 2021). More generally, persulfidation of transcription factors integrates hormonal and environmental signals by modulating DNA binding, protein stability, and protein–protein interactions. Crosstalk with NO-dependent Cys modifications adds an additional regulatory layer that refines stress signaling outputs (Aroca et al., 2015, 2017, 2018).

Connections between cysteine oxidation, reactive oxygen species, and persulfidation

A growing body of evidence supports a mechanistic coupling between Cys oxidation and persulfidation in plants (Filipovic et al., 2018; Zivanovic et al., 2019). Proteomic comparisons in Arabidopsis thaliana show that ∼5% of the proteome is persulfidated and that hundreds of proteins overlap with the sulfenylome, particularly proteins involved in stress responses, redox regulation, photosynthesis, and central metabolism (Zhou et al., 2020; Romero et al., 2026). Many proteins undergo both sulfenylation and persulfidation during oxidative stress, supporting the concept that ROS-mediated Cys oxidation primes proteins for persulfide formation (Filipovic et al., 2018; Zhou et al., 2020). This relationship is especially evident in photorespiration: when cellular H₂O₂ levels rise and the photorespiratory flux is suppressed, persulfidation of photorespiratory enzymes increases while sulfenylation decreases, highlighting a dynamic ROS–RSS interplay that stabilizes redox homeostasis (Aroca et al., 2023).

Functional studies across multiple plant pathways reinforce this oxidative priming model. In kinase signaling, oxidation of MPK4 at Cys181 is required for kinase activation and facilitates subsequent persulfidation, contributing to cold stress tolerance (Du et al., 2021). Similarly, reversible oxidation of cytosolic G6PDH under salt stress promotes persulfidation and protects the enzyme from irreversible overoxidation, preserving NADPH production required for antioxidant defense (Wang et al., 2023). Comparable mechanisms operate in glycolysis, where GAPDH undergoes cycles of oxidation, S-nitrosylation, and persulfidation that coordinate metabolic flux with cellular redox status (Bedhomme et al., 2012; Zaffagnini et al., 2013; Aroca et al., 2015).

Antioxidant enzymes provide additional plant-specific examples of ROS–persulfidation coupling. APX1, catalase, and class III peroxidases exhibit Cys modifications that alter activity in response to oxidative stress (Aroca et al., 2017; J. Li et al., 2020). H₂S-mediated persulfidation can enhance APX and peroxidase activity while differentially modulating catalase function, enabling rapid adjustment of antioxidant capacity and preventing irreversible oxidative damage (J. Li et al., 2020). Likewise, NADPH oxidases such as RBOHD, which are themselves ROS producers, are regulated by persulfidation, creating feedback loops that integrate ROS generation with sulfur signaling (Shen et al., 2020).

Redox-dependent Cys switches also regulate transcription in plants. S-nitrosylation and persulfidation modify bHLH, WRKY, and other transcription factors. These changes affect DNA binding, protein stability, and promoter selection, which shape stress-responsive gene expression (Aroca et al., 2017; Liu et al., 2019). These reversible Cys modifications allow plants to quickly adjust transcriptional networks during abiotic stress and hormonal signaling (Aroca et al., 2017, 2018). Similar mechanisms are found in mammals. For example, GAPDH and NF-κB p65 are persulfidated at redox-sensitive Cys residues under oxidative or nitrosative stress, showing that ROS–RSS crosstalk is evolutionarily conserved (Filipovic et al., 2018; Zivanovic et al., 2019).

Involvement of hydrogen sulfide in the responses to hypoxia

Hydrogen sulfide participates in O₂-dependent responses in animal cells

In mammalian cells, O₂ sensing is achieved by (de)stabilization of the dimeric transcription factor Hypoxia-Inducible Factor (HIF) which is dependent on O₂ availability (Ivan et al., 2001). Under normoxic conditions, one or both regulatory prolyl residues in HIF-α are hydroxylated by Prolyl Hydroxylase Domain (PHD) enzymes. The hydroxylated protein is recognized by the von Hippel–Lindau protein (pVHL) E3 ligase complex and targeted for proteasomal degradation. PHDs behave as O₂-sensing enzymes: physiological levels of hypoxia indeed inhibit PHD activity, allowing HIF-α to accumulate, dimerize with HIF-β, and activate transcription of genes involved in anaerobic metabolism, angiogenesis, erythropoiesis, and cell survival (Kaelin and Ratcliffe, 2008).

The first demonstration of other gaseous molecules involved in the modulation of the HIF/VHL pathway concerned NO (Berchner-Pfannschmidt et al., 2010). Early studies showed that high concentrations of NO can induce HIF-1α stability, probably by inhibiting the activity of PHDs, either by competition with O₂ at the ferrous iron (Fe²⁺) in the catalytic site of the enzymes or by reacting with Cys residues within the protein (Metzen et al., 2003; Chowdhury et al., 2011). More recently, a further mechanism of indirect regulation of O₂ sensing by NO has been identified at the level of 2-aminoethanethiol dioxygenase (ADO), a second O₂-sensing enzyme of humans with functional homology to oxygen-sensing plant cysteine oxidase (PCO) enzymes (Masson et al., 2019). NO can restrain ADO activity by affecting O₂ availability through inhibition of Complex IV, ultimately altering accumulation levels of ADO targets, such as the G-protein signaling protein 4 (RGS4) (Kim et al., 2025).

Increasing evidence also indicates that H₂S can modulate the HIF/pVHL O₂-sensing pathway (Fig. 3) (Peng et al., 2010; Olson, 2015; Wu et al., 2015). PHD2, the prominent PHD isoform in humans, contains two Cys residues that are reported to undergo persulfidation (Dey et al., 2020) at Cys21 and Cys33 (Dey et al., 2020); this persulfidation is essential for PHD2 association with HIF-1α and consequent hydroxylation. H₂S synthesis through CBS therefore promotes PHD2 activity and enhances HIF-1α hydroxylation, making it more susceptible to pVHL-mediated degradation. Notably, CBS expression was reported to decrease under hypoxia, suggesting that lower endogenous H₂S production may contribute to HIF-1α stabilization during O₂ deprivation (Dey et al., 2020). Thereby, CBS assumes an active role in monitoring O₂-deficiency responses in humans. However, the effects of H₂S on the hypoxic responses appear to be multifaceted. For example, exposure of Hep3B cells to 100 μM NaHS (an H₂S donor) has been shown to induce HIF-1α degradation under 1% O₂, but not under anoxia (0% O₂), indicating that O₂ may interact with H₂S, influencing its effect on HIF-1α (Kai et al., 2012). Additionally, H₂S can influence HIF-1α at the translational level. It has been demonstrated that exogenous NaHS suppresses hypoxia-induced HIF-1α accumulation by enhancing phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), resulting in reduced HIF-1α translation and decreased expression of downstream targets such as the vascular endothelial growth factor (VEGF) (Wu et al., 2012). This pVHL-independent inhibition reveals an additional downstream layer of control through which H₂S may modulate hypoxic signaling. Such conflicting observations may be attributed to differences in cell type, H₂S concentration, redox state, and experimental conditions (Budde and Roth, 2010; Lohninger et al., 2015; Dey et al., 2020).

Fig. 3.

Effects of H₂S on O₂ sensing in mammalian cells. Dotted lines represent the proposed effects of H₂S on O₂-related proteins and processes. H₂S produced by the CBS pathway can modulate the activity of HIF-α, both at the translational level and by increasing its degradation through enhancement of PHD activity via persulfidation. In addition, indirect repression of HIF-α activity may be associated with a modulation of mitochondrial O₂ levels, following the inhibition of Complex IV by H₂S. HIF-α stabilization and association with the HIF-β subunit promotes nuclear transcription under hypoxia. CBS, cystathionine β-synthase; HIF, hypoxia-inducible factor; pVHL, Von Hippel–Lindau tumor suppressor; PHD, prolyl-hydroxylase domain; SQR, sulfide:quinone oxidoreductase; ETHE1, ethylmalonic encephalopathy protein 1. Created in BioRender. Triozzi, P. (2026) https://BioRender.com/lr4cxu9.

Beyond mammalian cell culture systems, a role for H₂S in O₂ sensing is supported by evidence from multiple model organisms. In zebrafish embryos, silencing of CBS generates a hypoxia-like phenotype, which is recovered by exogenous NaHS supplementation, suggesting that endogenous H₂S contributes to low O₂ tolerance (Dey et al., 2020). In Caenorhabditis elegans, H₂S promotes stabilization of HIF-1α through a mechanism independent of pVHL, indicating a further unknown mechanism whereby H₂S intersects with O₂-sensing pathways, possibly through modulation of other redox-sensitive proteins (Budde and Roth, 2010).

H₂S can be produced and consumed by mitochondria, and its catabolism requires O₂ via the sulfide-oxidizing pathway, which involves enzymes such as sulfide:quinone oxidoreductase (SQR) (Jackson et al., 2012), ethylmalonic encephalopathy protein 1 (ETHE1) (Tiranti et al., 2009), and sulfite oxidase (Fu et al., 2025). Therefore, low O₂ conditions can limit mitochondrial H₂S oxidation, resulting in elevated intracellular levels (Marutani et al., 2021; Paul et al., 2021) (Fig. 3). H₂S accumulation can in turn protect cells by acting as a negative regulator of cytochrome c oxidase (Complex IV), thereby lowering O₂ consumption, reducing mitochondrial membrane potential, and decreasing ROS formation (Olson, 2015). These effects may support mitochondrial functions during O₂ scarcity by limiting energy demand and preventing oxidative damage. However, excessive H₂S accumulation, for instance due to deficiency in SQR or ETHE1 activity, results in the irreversible inhibition of Complex IV, which can lead to energetic deficiency even in the presence of O₂ (Tiranti et al., 2009). On the contrary, SQR expression improves mitochondrial respiration, maintains ATP production during hypoxia, and prevents sulfide-induced respiratory collapse. These findings suggest that the balance between H₂S production and oxidation is a key determinant of cellular adaptation to low O₂ levels (Marutani et al., 2021).

In adult mammals, the importance of H₂S in physiological O₂ sensing is most evident in the carotid bodies, small sensory organs that monitor arterial O₂ and regulate ventilation (Kumar and Prabhakar, 2012). Double knockout mice for CSE fail to increase H₂S levels in the carotid body during low O₂ exposure and exhibit severely impaired ventilatory responses; in addition, pharmacological inhibition of CSE produces similar effects (Peng et al., 2010). These findings indicate that CSE-derived H₂S is essential for the correct O₂ sensing responses.

Hydrogen sulfide in plant low O₂ responses

In plants, appropriate biosynthesis of H₂S underlies effective responses to low O₂ stresses, as demonstrated by lower submergence tolerance of the Arabidopsis H₂S-defective mutant des1 (Zhou et al., 2021; Telara et al., 2025, Preprint). However, a full understanding of the endogenous H₂S dynamics in tissues of plants exposed to low O₂ is still lacking. In waterlogged roots of Prunus persica (Xiao et al., 2020) and root tips of Pisum sativum (Cheng et al., 2013), as well as in root tips from submerged Zea mays seedlings (Peng et al., 2016), H₂S has been found to increase slightly as compared with control tissues after long-lasting treatments (24–72, 12–36, and 24–36 h, respectively). Submerged leaves of Arabidopsis can transiently accumulate H₂S in the first hours of stress, returning to basal levels after 12 h, according to two distinct studies where a comparable treatment set-up was applied (Yang et al., 2021; Xuan et al., 2022). However, no H₂S accumulation has been reported in Arabidopsis seedlings treated for a few hours with gaseous hypoxia (Telara et al., 2025, Preprint) or anoxia (Zhou et al., 2021), suggesting that physical entrapment of gaseous molecules is important to enable H₂S build-up. As stated before, most intracellular sulfur is not present as freely diffusible H₂S gas (its pKa being close to 7), but rather as hydrosulfide ion subjected to the acid trap mechanism. Nonetheless, lower diffusion of the volatile fraction may still contribute to the overall H₂S balance in submerged tissues.

H₂S accumulation under stress is generally accompanied by elevated expression of cysteine desulfhydrase genes (Fig. 1). While this is well documented in several stresses, including drought, salinity, cold, oxidative stress, and ABA treatment, as well as during bacterial pathogen infection (Vojtovič et al., 2021), few studies have investigated the transcriptional regulation of H₂S biosynthetic genes under low oxygen and, surprisingly, none has revealed DES1 up-regulation (Yang et al., 2021; Xuan et al., 2022). H₂S steady state is the result of biosynthesis and consumption, due to H₂S incorporation into Cys by the action of cysteine synthase complexes, reactions with oxidized sulfur compounds, and formation of persulfide groups in proteins (Romero et al., 2014; Aroca et al., 2018; Filipovic et al., 2018). Lack of SQR homologous sequences in Arabidopsis (Theissen et al., 2003) makes the occurrence of the canonical mitochondrial sulfide oxidation pathway found in animals (Fig. 3) uncertain. The block of mitochondrial sulfide oxidation, serving as a main mechanism for H₂S induction under hypoxia in animals, is likely to be replaced in plants by the regulation of other processes in sulfur metabolism (sulfate assimilation, GSH metabolism, mitochondrial sulfur catabolism under starvation through ETHE1, and Cys synthesis) (Krüßel et al., 2014) that alter H₂S sinks and ultimately its steady state under low oxygen. Differences in global persulfidation might also account for the discrepancy observed between hypoxic seedlings (Telara et al., 2025, Preprint), where H₂S levels decreased, and those treated with anoxia by Zhou et al. (2021), where no decrease was reported. Both conditions generate ROS (Pucciariello and Perata, 2017), but anoxia may elicit a different ROS production profile, determining lower availability of oxidized protein targets for H₂S interaction than hypoxia, and thereby resulting in a weaker H₂S sink. Higher ROS generation in anoxia is indeed likely to push protein thiol oxidation to Cys sulfinic/sulfonic acid, rather than to the Cys sulfenic state that can support persulfidation.

Discrepancies in H₂S content recorded in the literature might also be reflecting tissue-specific dynamics in H₂S regulation, making the comparison of different experimental designs misleading. For example, Zhou et al. (2021) analyzed free H₂S in the roots of A. thaliana, whereas Telara et al. (2025, Preprint) focused on the whole seedlings. H₂S content has been mainly determined in whole tissue extracts, using the methylene blue method (Cheng et al., 2013; Xiao et al., 2020; Yang et al., 2021; Xuan et al., 2022), or whole seedlings (via GC-MS/MS; Telara et al., 2025, Preprint); occasionally, it has been revealed by in situ detection of the H₂S-reactive fluorescent probe WSP-1 (Peng et al., 2016; Yang et al., 2021; Zhou et al., 2021). Techniques characterized by higher spatial resolution (such as the use of microelectrodes or genetically encoded reporters for H₂S) may further disclose the existence of tissue-specific regulation of H₂S production under stress, which is presently unknown.

The possibility of modulating plant responses to low O₂ stresses by acting on their H₂S levels has been explored in Arabidopsis and a few other species, where evidence has been gathered that pre-treatments with the H₂S donor NaHS can protect plants against subsequent submergence or waterlogging stress (Table 1). H₂S appears to be implicated in plant low O₂ physiology at different levels, by regulating relevant signaling, stress response, and metabolic processes (Fig. 4A). The activation of antioxidant defenses appears to be a common mechanism underlying the beneficial effects of H₂S on plant survival to different conditions, ranging from heavy metal poisoning, temperature stress, salinity, to drought (reviewed in Zhou et al., 2025). This has been shown to occur through the regulation of either antioxidant gene expression or the levels of redox metabolites (Birke et al., 2015), or through persulfidation of relevant stress-associated enzymes (Aroca et al., 2015). However, a description of stress-specific mechanisms is generally not available. Under submergence or waterlogging, NaHS pre-treatments enhance the activity of ROS-scavenging enzymes such as superoxide dismutase, catalase, and APX, GSH synthesis, and the activity of alternative oxidase in mitochondria (Cheng et al., 2013; Peng et al., 2016; Xiao et al., 2020; Zhong et al., 2023). The increased antioxidant capacity can in turn alleviate the stress-induced death of root tips, a ROS-sensitive tissue.

Table 1.

Overview of the impact of exogenous NaHS treatments on plant responses to low O₂ stresses

Low oxygen stress	H2S treatment	Effect on stress response	Growth stage	Species	Reference
Dark submergence	Pre-treatment performed by 1 h 100 µM NaHS misting every 12 h for 3 d	Increased survival rate	Adult plants	A. thaliana	Yang et al. (2021)
Dark submergence	Pre-treatment performed by 1 h 100 µM NaHS misting every 12 h for 3 d	Increased survival rate, increased membrane integrity leaves	Adult plants	A. thaliana	Xuan et al. (2022)
Dark submergence	Fumigation with 50 µM NaHS solution for 24 h before the stress	Decreased root tip death, decreased ROS accumulation	Seedlings	A. thaliana	Xuan et al. (2022)
Dark submergence	Pre-treatment with 100 µM NaHS spraying (1 ml) every 12 h for 1.5 d	Increased survival rate	Adult plants	A. thaliana	Zhou et al. (2021)
Waterlogging under photoperiod 15 h light/9 h dark	Pre-treatment performed by transfer to 20–600 µM NaHS Hoagland solution for 1 h every 12 h for 1.5 d (best effect at 100 µM)	Alleviation of hypoxia- and re-oxygenation-induced root tip death, lower ROS accumulation in root tips, decreased ethylene accumulation	Seedlings	Pisum sativum	Cheng et al. (2013)
Submergence under light condition	Pre-treatment performed by transfer to 20–1000 µM NaHS Hoagland solution for 1.5 h every 12 h for 2 d (best effect at 100 µM)	Alleviation of hypoxia-induced root tip death, stimulation of antioxidant systems	Seedlings	Zea mays	Peng et al. (2016)
Waterlogging under photoperiod 15 h light/9 h dark	20–300 µM NaHS supplementation in water at the onset of the stress (best effect at 200 µM)	Alleviation of hypoxia-induced root tip death, decreased ethylene accumulation in roots, decreased ROS accumulation in roots and leaves	Seedlings	Prunus persica	Xiao et al. (2020)
Waterlogging under 12 h of light per day	10–1000 µM (best effect at 100 µM and 200 µM)	Decreased ROS accumulation, induction of Ca2+ influx, decreased mitochondrial damage, activation of alternative respiratory pathways in roots	Seedlings	Avicennia marina	Zhong et al. (2023)

Submergence refers to complete flooding of the plants, and waterlogging to root flooding.

Fig. 4.

Involvement of H₂S as a gasotransmitter during flooding-associated stresses. (A) Summary of mechanisms of H₂S signaling under submergence or waterlogging. Proposed regulatory roles include ROS scavenging, crosstalk with hormone pathways, interactions with autophagy, and the modulation of PCO-dependent O₂ sensing through protein persulfidation (-SSH). Dotted lines represent multistep or indirect processes. Hypothetical mechanisms are indicated by question marks. (B) The cysteine N-degron pathway (Cys-NDP) mediates the conditional degradation of the ERFVII transcription factors in the presence of oxygen and nitric oxide (above) via the sequential action of PCO, ATE, and PRT6 enzymes. Hypoxia prevents PCO action, thereby stabilizing the ERFVII factors and enabling hypoxic gene expression (below). *Cys, oxidized cysteine; APX, ascorbate peroxidase (AT1G07890); ATG18a, autophagy-related protein 18a (AT3G24220); ATE, arginyl-tRNA protein transferase (ATE1: AT5G05700; ATE2: AT5G63280); CAT, catalase (AT4G35090); CoASH, coenzyme A; ERFVII, ethylene response factor VII (RAP2.12: AT1G53910, RAP2.2: AT3G14230, RAP2.3: AT3G16770, HRE1: AT1G72360, HRE2: AT2G40340); ERO, endoplasmic reticulum oxidoreductin (ERO1: AT1G15100, ERO2: AT2G25660); HRG, hypoxia-responsive genes; HRPE, hypoxia-responsive promoter element; PCO, plant cysteine oxidase (PCO1–PCO5: AT5G15120, AT1G05160, AT3G03040, AT4G36940, AT2G44810); PRT6, E3 ubiquitin ligase proteolysis 6 (AT5G02310); RBOHD, respiratory burst oxidase homolog D (AT5G47910); SOD, superoxide dismutase (AT1G08830). Created in BioRender. Giuntoli, B. (2026) https://BioRender.com/yupkfkj.

The involvement of H₂S in the regulation of autophagic processes appears to extend to submergence conditions. Autophagy is essential to plant survival under submergence (Chen et al., 2015; Guan et al., 2019; Yang et al., 2025), and its promotion has been indicated as a mechanism co-occurring with the improved submergence tolerance of H₂S-pre-treated plants (Xuan et al., 2022). Therefore, while sulfide represses autophagy triggered by carbon starvation in normoxic conditions (Álvarez et al., 2012b; Aroca and Gotor, 2022), it instead exerts pro-autophagy effects in submergence conditions, suggesting that, under low O₂ conditions, H₂S signaling interacts with distinct mechanisms of carbon management. Additional mechanisms might involve interactions between H₂S, autophagy, and low O₂-induced ER stress. Different stressors, including hypoxia, can cause the accumulation of misfolded proteins in the ER, leading to the onset of ER stress conditions (Pereira et al., 2014; Bartoszewska and Collawn, 2020; Ugalde et al., 2022) and the activation of protective unfolded protein response (UPR) pathways. In mammals, the involvement of H₂S in the UPR has been reported (Krishnan et al., 2011). Remarkably, in plants, the UPR is regulated by O₂-dependent endoplasmic reticulum oxidoreductin (ERO) 1/2 enzymes, whose mutation in Arabidopsis impairs survival upon low O₂ (Zhou et al., 2021; Ugalde et al., 2022). On the other hand, in normoxic conditions, ER stresses can trigger ER degradation through autophagy (also called ER-phagy), with the participation of H₂S signaling acting through ATG18a persulfidation (Aroca et al., 2021). While mechanistic evidence is missing, these separate elements suggest that H₂S signaling might be connected with hypoxia-activated UPR pathways and ER-phagy in plants (Fig. 4A).

Pre-treatment with NaHS causes changes in amino acid metabolism, hormone production, and signal transduction taking place during subsequent submergence. The interplay between H₂S and hormone regulation under low O₂ has not been extensively characterized. Evidence of repression of cytokinin- and auxin-dependent signaling emerges from transcriptomic data in Arabidopsis (Yang et al., 2021). Moreover, it has been proposed that H₂S might improve plant defense and antioxidant responses in the desubmergence phase through the activation of jasmonic acid-dependent signaling (Yuan et al., 2017; Yang et al., 2021). Interestingly, H₂S has been found to participate in a negative feedback regulation with ethylene, the major flooding-related hormone. H₂S can act downstream of ethylene during stress responses, mediating part of the protective effects of ethylene, such as in the case of ethylene-induced stomatal closure, while also down-regulating ethylene biosynthesis (Liu et al., 2012; Hou et al., 2013; Jia et al., 2018). Also in waterlogged roots, H₂S pre-treatment has been found to counteract ethylene accumulation (Cheng et al., 2013; Xiao et al., 2020). Although vastly implied by the literature (Liu et al., 2021), the interactions between H₂S and hormone-dependent physiological phenomena are supported by surprisingly few mechanistic links. These encompass persulfidation of SnRK2.6 and RBOHD in guard cells (Chen et al., 2020; Shen et al., 2020), and of the ethylene biosynthetic enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase of Solanum lycopersicum during exposure to osmotic stress (Jia et al., 2018). The latter modification may underlie ethylene down-regulation during waterlogging and, potentially, regulate stomatal closure responses in this condition. The network of gaseous signals that crosstalk under low O₂ conditions further extends to NO. Ethylene, which is known to modulate NO levels under hypoxia through phytoglobins (Hartman et al., 2019), might interfere with downstream H₂S signaling indirectly through NO–H₂S synergy.

Chemical interventions with H₂S donors and the use of des1 mutants indicate that H₂S is essential to the activation of hypoxia-inducible genes following the onset of low O₂ conditions. In Arabidopsis, NaHS supplementation boosts hypoxia marker gene expression, whereas defective production of endogenous H₂S associates with lower gene induction (Yang et al., 2021; Xuan et al., 2022; Telara et al., 2025, Preprint). Hypoxic transcription is coordinated by ERFVII (Ethylene Responsive Factor group VII) family transcription factors, which activate the expression of hypoxia-responsive genes by direct association with conserved cis-elements in their promoters (Gasch et al., 2016) (Fig. 4B). In turn, the ERFVII proteins are directly regulated by O₂ availability through a mechanism of conditional destabilization similar to that impinging on HIF-1α in humans. In the presence of O₂, PCO enzymes oxidize the conserved ERFVII residue Cys2, thereby funneling these factors into a dedicated pathway of proteasomal degradation, the Cys/Arg N-degron pathway (Weits et al., 2014). Conversely, lack of O₂ inhibits PCOs and enables ERFVII accumulation, triggering the hypoxic response (Fig. 4B).

In this context, H₂S has been shown to inhibit PCO activity in Arabidopsis, revealing that H₂S participates in the induction of hypoxia-responsive genes by contributing to ERFVII stabilization (Telara et al., 2025, Preprint). Persulfidation has been detected on recombinant AtPCO4 upon H₂S supplementation and, in vivo, in hypoxia-treated Arabidopsis seedlings (Telara et al., 2025, Preprint). Moreover, persulfidation of AtPCO3 has been reported by Aroca et al. (2017) in unstressed wild-type leaves. The occurrence of this PTM in the regulation of human and plant O₂-sensing enzymes (namely PHDs and PCOs), although apparently deployed for opposite outcomes (Figs 3, 4), reinforces the relevance of H₂S as a gasotransmitter under low O₂. PCOs are also inhibited in the presence of H₂O₂ (Akter et al., 2026) and potentially affected by S-nitrosylation, as shown in the case of PCO3 (Hu et al., 2015). Besides their role as O₂ sensors, PCOs thus appear to serve as a convergence point of ROS- and NO-mediated signaling pathways under hypoxia. It is tempting to speculate on the participation of H₂S in the same mechanism, with PCO persulfidation occurring on catalytically relevant Cys residues previously targeted by oxidation or S-nitrosylation. Evidence for this scenario, and its implications for plant hypoxia physiology, will emerge from experiments aimed at disentangling the mechanisms of action of the signaling molecules involved in PCO enzyme regulation.

Conclusions and perspectives

Historically regarded as a toxic metabolic byproduct, H₂S has now been identified as the third gasotransmitter molecule in plants, after NO and CO, with wide-ranging beneficial effects. Recent studies have revealed its fundamental role in the regulation of plant development and the integration of plant defense responses against various abiotic stresses including drought, salinity, and flooding. Despite the heterogeneous experimental approaches adopted in the literature, an emerging consensus suggests that submergence tolerance is influenced by H₂S levels. The beneficial effects of H₂S have been traced back to its manyfold intersections with hypoxia-associated pathways of ROS management, ER signaling, autophagy, and O₂ sensing. Moreover, H₂S is now known to take part in signaling that involves the other two main gaseous signals implicated in plant hypoxic regulation, namely ethylene and NO.

H₂S exerts its roles mainly through protein persulfidation. So far, direct hypoxic regulation of protein functions via persulfidation has only been demonstrated for PCO O₂-sensing enzymes. PCO inhibition by H₂S affects the stability of ERFVII transcription factors and the induction of hypoxia-responsive genes, ultimately influencing plant metabolic adaptations to low O₂. However, hypoxia is likely to have a more widespread impact on protein persulfidation profiles, considering the effects of low O₂ on NO levels and redox balance and their connections with H₂S-dependent PTMs. Efforts aimed at the characterization of the persulfidome of plants exposed to O₂ deprivation will provide relevant insights on the impact of H₂S signaling under hypoxia.

The application of H₂S to crops, potentially in combination with its synergistic partner NO, is considered as a viable protective strategy against abiotic stresses (Dos Reis et al., 2026). Crop priming with H₂S-based formulations might be especially useful for high-value cereals such as barley or wheat, which suffer from severe yield losses due to flooding. Crop treatments where the active component is an endogenous gasotransmitter, rather than an ‘unnatural’ agrochemical, may be favorably adopted by farmers and consumers. The development of effective agricultural solutions derived from H₂S administration is, however, still hindered by scant understanding of the tissue- and dose-dependent responses, in addition to known H₂S dose-dependent toxicity effects. Although at low concentrations it plays a positive signaling role, at higher concentrations H₂S can trigger toxicity by impairing mitochondrial function. Defining thresholds at which the transition from positive to negative function occurs in specific relevant crops is crucial to enable effective flooding protection strategies based on H₂S application or crop genetic improvement. For the same reason, the choice of H₂S donor compounds and the release kinetics of H₂S from any formulation (Dos Reis et al., 2026) are expected to be crucial aspects in the development of H₂S-based supplements for agriculture.

Author contributions

SA, EF, BG, and YT: conceptualization, writing—original draft, and writing—review & editing; EF and BG: funding acquisition and supervision; SA and YT: visualization.

Funding

BG and YT were supported by the Italian Ministry of University and Research with grant nos FIS-2023-00430 and PRIN-2022-RBHRJR. SA and EF were supported by the European Research Council (European Union’s Horizon 2020 Research and Innovation Programme grant no. 864888).