Reactive Oxygen and Carbonyl Species: Dual Regulators of Abiotic Stress Signaling and Tolerance in Plants

Abstract

Reactive oxygen species (ROS) are integral components of plant signaling networks that mediate interactions between plants and their environment, thereby regulating diverse physiological and biochemical processes. While controlled ROS production is essential for stress perception and signal transduction, excessive ROS accumulation induces oxidative damage. ROS-mediated lipid peroxidation of polyunsaturated fatty acids leads to the formation of highly electrophilic α,β-unsaturated carbonyl compounds collectively referred to as reactive carbonyl species (RCS). Under severe abiotic stress conditions, excessive RCS accumulation exerts cytotoxic effects and causes widespread cellular dysfunction. In contrast, at subtoxic levels, RCS function as important secondary messengers that modulate stress-responsive signaling pathways, including programmed cell death, stomatal regulation, and adaptive responses to abiotic stresses. This review critically synthesizes current advances in understanding the dual roles of ROS and RCS as both damaging agents and signaling molecules in plants. Particular emphasis is placed on the mechanistic basis of ROS-RCS crosstalk and their interactions in abiotic stress tolerance. Furthermore, this review highlights emerging research gaps and outlines future perspectives aimed at translating redox signaling insights into strategies for improving plant stress resilience under changing environmental conditions.

1. Introduction

Abiotic stresses such as drought, salinity, extreme temperatures, heavy metal toxicity, and flooding negatively affect plant survival, growth, and productivity [1,2]. Abiotic stress disrupts water uptake, nutrient balance, enzyme activation, cellular homeostasis, gene expressions, and hormonal signaling in plants, leading to decreased photosynthesis, stunted growth, and reduced yield [3,4]. It has been reported that abiotic stresses are estimated to reduce global crop productivity by 50–80% relative to optimal conditions, with drought and salinity being the most economically significant constraints [5].

To survive under adverse abiotic conditions, plants have evolved sophisticated adaptive mechanisms involving complex signaling networks. Once regarded solely as toxic byproducts of aerobic metabolism, reactive oxygen species (ROS) are now recognized as central regulators of stress perception and adaptive responses. At low levels, ROS act as signaling molecules that mediate stress perception, activate defense-related gene expression, and regulate key developmental processes [6,7]. In this context, ROS are known to modulate stomatal movement, root architecture, and phytohormone signaling, thereby linking environmental cues to coordinated physiological responses in plants [8,9].

In contrast, excessive or uncontrolled ROS accumulation leads to oxidative damage of lipids, proteins, nucleic acids, and carbohydrates, ultimately disrupting cellular homeostasis and potentially causing irreversible metabolic dysfunction and plant death [10,11]. Thus, the functional outcome of ROS signaling, beneficial or deleterious, depends critically on their spatiotemporal distribution, concentration thresholds, and cellular compartmentalization, underscoring the fine balance between ROS-mediated signaling and oxidative stress in plants [12]. Under stress, plant cells continuously produce ROS, which can oxidize membrane lipids, particularly polyunsaturated fatty acids, and form lipid peroxides. These peroxides can further break down into carbonyl compounds such as aldehydes, ketones and hydroxy acids by enzymatic or non-enzymatic reactions [13,14]. The aldehydes and ketones with α,β-unsaturated carbonyls groups are called reactive carbonyl species (RCS) because of their high electrophilicity and reactivity [14]. The excessive accumulation of RCS under severe abiotic stresses is toxic to plants and can cause extensive cellular damage [14,15,16]. Excessive levels of aldehydes, a major class of RCS, disrupt membrane integrity, protein function, and cellular homeostasis, leading to severe cellular injury [17]. In contrast, at optimal concentrations, RCS function as critical signaling molecules in plant stress responses, including the initiation of programmed cell death (PCD), regulation of stomatal movement, activation of antioxidant defense systems, and induction of senescence [17,18,19].

Notably, low levels of malondialdehyde (MDA), a representative RCS, have been shown to induce the expression of key abiotic stress-responsive genes, including those associated with oxidative stress, drought, and heat shock responses in Arabidopsis thaliana, providing strong evidence for a signaling role of RCS in stress acclimation [17,20]. Beyond stress responses, ROS-RCS crosstalk also regulates developmental processes. Recent studies further indicate that RCS act downstream of ROS production and contribute to the fine-tuning of auxin signaling during lateral root (LR) formation. In this context, RCS serve as a feed-forward signaling link between ROS and auxin pathways, amplifying auxin responses initiated by ROS signals [21].

Together, ROS and RCS constitute a dynamic and interconnected redox signaling network, in which ROS act as primary oxidative signals, while RCS function as secondary amplifiers that translate oxidative cues into precise physiological and transcriptional responses. This coordinated ROS–RCS signaling system is particularly crucial under abiotic stress conditions, where redox imbalance poses a major threat to plant survival while simultaneously activating adaptive signaling pathways. Although previous reviews have comprehensively discussed reactive species networks [22,23,24], often integrating ROS with other molecules such as RNS and RSS, a detailed mechanistic dissection of ROS–RCS interactions is still lacking. Moreover, while ROS signaling [22] and RCS toxicity [14] have been extensively examined separately, their integrated crosstalk and coordinated regulatory roles in plant stress responses have not been systematically resolved. In this review, we synthesize current knowledge on the dual roles of ROS and RCS as both damaging agents and indispensable signaling molecules in plant abiotic stress tolerance. We further highlight critical knowledge gaps and propose future research directions aimed at exploiting ROS–RCS-mediated redox networks for improving crop resilience and advancing sustainable agricultural practices.

2. ROS and RCS Production in Plants

2.1. Generation of ROS in Plants

Reactive oxygen species are partially reduced forms of molecular oxygen. Due to the ground-state triplet configuration of ¹O₂, direct four-electron reduction to water is spin-forbidden, resulting in stepwise single-electron transfers that generate superoxide (O₂•⁻), hydrogen peroxide (H₂O₂), and hydroxyl radical (OH•) [25]. It is well-known that ROS are constitutively generated in almost all subcellular compartments of plant cells. In photosynthetic tissues, chloroplasts and peroxisomes represent major sources of ROS under light conditions, whereas mitochondria contribute predominantly in non-photosynthetic tissues [26,27]. ROS are produced in both non-stressed and stressed cells across multiple cellular compartments, including chloroplasts, mitochondria, peroxisomes, plasma membranes, endoplasmic reticulum, apoplast, and cell walls (Figure 1) [26]. The rate and nature of ROS generation vary depending on plant tissue, developmental stage, and environmental conditions [28]. Although the mechanisms of ROS production in plants are well documented [26,28], this section briefly summarizes the major sites of ROS generation. We first provide an overview of ROS generation sites to establish the context for understanding ROS-RCS crosstalk in subsequent sections.

Chloroplasts are primary ROS sources in photosynthetic tissues. Singlet oxygen (¹O₂) forms via energy transfer from excited chlorophyll, while superoxide (O₂•⁻) arises from direct electron leakage at multiple sites in the photosynthetic electron transport chain, including PSI (Mehler reaction), PSII, and the cytochrome b₆f complex [11]. Superoxide is rapidly converted to H₂O₂ by superoxide dismutase, and under stress conditions, H₂O₂ can generate hydroxyl radicals (OH•) via Fenton chemistry [29]. Stomatal closure and reduced CO₂ fixation during stress further enhance chloroplastic ROS production by over-reducing the electron transport chain [30].

Mitochondria serve as major ROS sources in non-photosynthetic tissues and during darkness. Electron leakage from Complexes I and III of the respiratory chain produces O₂•⁻, which is converted to H₂O₂ by mitochondrial superoxide dismutase [31]. In plant mitochondria, Complex I contributes to superoxide production primarily during reverse electron transport under conditions of high membrane potential, while Complex III generates superoxide on both the matrix and intermembrane space sides [32]. Superoxide is rapidly converted to H₂O₂ by antioxidant enzymes, and H₂O₂ may subsequently form OH• under stress conditions [11].

In addition, peroxisomes are important contributors to cellular ROS homeostasis; in the peroxisomes, O₂ accepts electrons during various oxidative reactions. Glycolate oxidase accounts for approximately 70% of peroxisomal H₂O₂ production [33]. In addition to H₂O₂, peroxisomes and glyoxysomes generate O₂•⁻ and ¹O₂, particularly during seed germination [32,34].

Additionally, other cellular compartments also contribute to ROS generation. In the endoplasmic reticulum, NAD(P)H-dependent electron transport involving cytochrome P450 enzymes produces O₂•⁻ [35]. Plasma membrane-localized oxidoreductases and NADPH oxidases generate ROS in response to developmental and stress signals [36,37]. In addition to these, ROS production in the apoplast and cell wall is mediated by peroxidases and other oxidases, contributing to cell wall remodeling and defense responses [38].

The biological significance of ROS depends critically on their subcellular localization and local concentration. While basal ROS levels in unstressed cells are typically maintained at 0.1–1 μM for H₂O₂ and low nanomolar for O₂•⁻ and OH•, stress-induced ROS bursts can transiently elevate concentrations 10- to 100-fold in specific microdomains [39]. This spatiotemporal specificity enables ROS to function as localized signals rather than merely diffusible damage agents.

2.2. Generation of RCS in Plants

Reactive carbonyl species are a class of oxylipins characterized by α,β-unsaturated carbonyl structures [14]. In plants, RCS are primarily generated through the oxidative degradation of lipid hydroperoxides (LOOHs) (Figure 1) [14]. Lipid hydroperoxides originate from both enzymatic oxygenation of membrane lipids and non-enzymatic oxidation of polyunsaturated fatty acids (PUFAs) mediated by ROS [40]. The formation of RCS via enzymatic and non-enzymatic pathways has been well documented in the literature [14,40]. In the enzymatic pathway, lipoxygenases (LOXs) play a central role by catalyzing the dioxygenation of PUFAs, leading to the formation of LOOHs [41,42]. In contrast, non-enzymatic lipid peroxidation has been largely elucidated through in vitro studies on oil deterioration under thermal and oxidative conditions [43]. As abiotic stresses predominantly enhance ROS production and lipid peroxidation, this section briefly emphasizes the non-enzymatic pathways of RCS formation.

During non-enzymatic lipid peroxidation, highly reactive ROS generated in close proximity to cellular membranes initiate lipid oxidation by abstracting a hydrogen atom from the pentadiene structure of PUFAs. The resulting lipid radical rapidly reacts with molecular oxygen to form a lipid peroxyl radical, which in turn propagates radical chain reactions by oxidizing neighboring lipid molecules, ultimately leading to extensive lipid peroxidation [44]. In addition to radical-mediated reactions, the direct addition of singlet oxygen (¹O₂) to PUFAs also contributes to LOOH formation [45]. Subsequent enzymatic and non-enzymatic decomposition of LOOHs gives rise to a diverse array of oxylipin-derived carbonyl compounds, including RCS with varying carbon-chain lengths and degrees of oxygenation [20,46]. Major plant RCS include acrolein, malondialdehyde (MDA), 4-hydroxy-(E)-2-nonenal (HNE), (E)-2-pentenal, (E)-2-hexenal, crotonaldehyde, 4-oxo-2-nonenal, 4-hydroxy-(E)-2-hexenal (HHE), and β-cyclocitral [14].

The spectrum of RCS produced via non-enzymatic lipid peroxidation is complex and depends on the PUFA substrate, initiating ROS, and local microenvironment. Linoleic acid (18:2) peroxidation predominantly yields 4-hydroxy-(E)-2-nonenal (HNE) and shorter-chain aldehydes, while α-linolenic acid (18:3) generates 4-hydroxy-(E)-2-hexenal (HHE) and acrolein [47]. Under severe oxidative stress, secondary reactions of RCS with proteins and other lipids can generate more complex carbonyl-containing products, including advanced lipoxidation end products (ALEs), further amplifying cellular damage.

The biological reactivity of RCS derives from their electrophilic α,β-unsaturated carbonyl structure, in which the carbonyl group withdraws electron density from the β-carbon, rendering it susceptible to nucleophilic attack (Michael addition). The reactivity hierarchy follows: 4-oxo-2-alkenals > 4-hydroxy-2-alkenals > 2-alkenals > saturated aldehydes [48]. Among plant RCS, acrolein is the most reactive but least specific, reacting rapidly with diverse nucleophiles. In contrast, HNE exhibits intermediate reactivity with greater selectivity for protein thiol groups, particularly cysteine residues in structured proteins where local environment modulates reactivity. This differential reactivity suggests that distinct RCS may convey different signals or target different protein populations, although this hypothesis requires systematic testing.

3. Role of ROS and RCS in Abiotic Stress Responses

3.1. Dual Role of ROS in Abiotic Stress Signaling

ROS functions as both signaling molecules and damaging agents are fundamentally determined by concentration thresholds, temporal dynamics, and subcellular localization [26]. It has been reported that basal ROS levels (typically <100 nM H₂O₂ in the cytosol) participate in redox regulation of metabolic enzymes and maintain cellular homeostasis [39] under non-stress conditions. However, transient elevation to low micromolar concentrations (1–10 μM H₂O₂) activates specific signaling pathways through reversible oxidation of redox-sensitive cysteine residues in target proteins, including receptor kinases, phosphatases, and transcription factors [12].

In contrast, excessive ROS accumulation (>50–100 μM) overwhelms antioxidant defenses and causes oxidative damage to lipids, proteins, and nucleic acids [49]. This concentration-dependent switching is exemplified during stomatal closure, where localized H₂O₂ production in guard cells reaches 5–10 μM, sufficient to activate Ca²⁺ channels and inhibit K⁺ influx channels, but below the threshold for oxidative damage [50]. Similarly, H₂O₂ gradients of 1–5 μM regulate cell elongation and differentiation in root tip cells, while concentrations exceeding 20 μM inhibit root growth and trigger cell death [51].

In addition, the temporal dimension is equally critical. Transient ROS bursts lasting minutes to hours typically function as signals, whereas chronic elevation over days leads to cumulative damage. Importantly, during pathogen attack, the oxidative burst plays a direct antimicrobial role, contributing to the inhibition or killing of invading pathogens through the rapid accumulation of toxic ROS at the infection site. At the same time, pathogen-triggered ROS bursts are tightly controlled in duration and magnitude through the coordinated activation of NADPH oxidases and antioxidant systems, thereby minimizing collateral damage while also enabling effective defense signaling [52]. Under abiotic stress, prolonged ROS production can shift from adaptive to maladaptive, necessitating enhanced antioxidant capacity to restore signaling precision.

3.2. ROS-Mediated Mechanisms and Plant Responses Across Abiotic Stresses

Despite differences in the primary stress stimulus, ROS-mediated adaptive responses share common mechanistic elements across stress types. First, ROS act as rapid local signals that trigger immediate protective responses, such as stomatal closure during drought or salinity, membrane stabilization during cold, and protein protection under heat [53]. Second, ROS initiate systemic signaling through ROS waves cell-to-cell propagation of oxidative signals that prepare distal tissues for impending stress, a phenomenon termed systemic acquired acclimation (SAA) [54]. Third, ROS-activated MAP kinase cascades converge on transcriptional reprogramming, inducing overlapping sets of stress-responsive genes including antioxidants, heat shock proteins, and osmolyte biosynthesis enzymes [55].

Additionally, ROS modulate phytohormone signaling networks, particularly ABA, jasmonic acid, and ethylene, creating integrated stress response networks [56]. Finally, ROS-mediated Ca²⁺ signaling represents a universal stress transduction mechanism, with stress-specific Ca²⁺ signatures decoded by calcium-dependent protein kinases (CDPKs/CPKs) that activate distinct downstream pathways [57]. These shared mechanisms provide a framework for understanding stress-specific adaptations.

Drought is a major abiotic stress that severely hampers plant growth and development [58]. Numerous studies have reported that drought stress induces production of ROS in plants, where ROS play a primary role in regulating water loss and stress adaptation [59,60]. In particular, a moderate increase in H₂O₂ levels mediated by abscisic acid (ABA), acts as a key signaling component that triggers stomatal closure and regulates gas exchange [61]. Beyond local signaling, ROS waves can propagate systemically from drought-affected tissues to distal organs, leading to the pre-activation of defense responses such as antioxidant enzyme systems and osmolyte accumulation. This long-distance ROS signaling establishes a “primed” physiological state that enhances whole-plant tolerance to drought stress [54,62].

In salinity stress, ROS signaling plays a critical role in maintaining cellular ion homeostasis. The H₂O₂ activates components of the SALT OVERLY SENSITIVE (SOS) pathway, which is essential for the extrusion of excess sodium ions from the cytosol and for limiting Na⁺ toxicity [63]. Concurrently, ROS modulate root system architecture by influencing cell wall dynamics and Ca²⁺-dependent signaling pathways, enabling plants to restrict sodium uptake and adapt root growth toward less saline soil regions [64].

During heat stress, a transient and acute burst of ROS, originating primarily from mitochondria and chloroplasts, is sensed and transduced through mitogen-activated protein kinase (MAPK) signaling pathways, which activate heat shock factors (HSFs). Activated HSFs subsequently induce the expression of heat shock proteins (HSPs), a class of molecular chaperones that protect cellular proteins from denaturation and aggregation under high-temperature conditions [65,66]. This ROS–HSP signaling axis constitutes a fundamental component of acquired thermotolerance in plants [67].

Under cold stress conditions, a more sustained and low-intensity ROS signal plays a critical regulatory role. H₂O₂ acts upstream of the C-repeat binding factor (CBF) transcriptional cascade, a central regulator of cold acclimation [68]. Through ROS-mediated signaling, plants enhance the expression of genes involved in fatty acid desaturation to preserve membrane fluidity and activate antioxidant defense systems that protect the photosynthetic machinery from cold-induced photo-oxidative damage [69].

Under heavy metal stress, ROS acts as a key signaling molecule that conveys stress information to the nucleus, thereby promoting the transcriptional activation of genes involved in metal detoxification, including those encoding metal-chelating peptides such as phytochelatins and metallothioneins [70]. ROS signaling enhances the expression of membrane transporters responsible for vacuolar sequestration of metal ions, effectively reducing their cytosolic concentrations and mitigating toxicity [71]. Beyond detoxification, ROS-dependent signaling also regulates broader adaptive responses under heavy metal stress, including the activation of mitogen-activated protein kinase (MAPK) cascades, modulation of Ca²⁺ signaling, and induction of antioxidant defense genes. For example, H₂O₂-mediated signaling has been shown to activate MAPKs that coordinate metal stress responses, while simultaneously inducing enzymatic antioxidants such as SOD, CAT, and APX to maintain redox homeostasis [72]. Collectively, these responses exemplify a tightly regulated feedback mechanism in which heavy metal-induced ROS production simultaneously triggers ROS-dependent signaling pathways that activate detoxification, antioxidant defense, and cellular homeostasis, thereby limiting metal-induced oxidative damage [73].

3.3. Dual Role of RCS in Signaling, Toxicity, and Abiotic Stress Responses

Recent studies demonstrate that RCS act both as cytotoxic by-products and as multifunctional signaling intermediates within the ROS–lipid peroxidation network that underpins plant stress physiology. Due to their electrophilic nature, RCS readily react with nucleophilic cellular constituents and become highly deleterious when they accumulate to excessive levels, causing widespread cellular toxicity [19]. In contrast, at optimal concentrations, RCS function as important signaling molecules that regulate diverse stress-responsive processes, including PCD, stomatal movement, antioxidant defense activation, and senescence [17,18,19]. This dual functionality positions RCS along a continuum ranging from adaptive signaling mediators to potent cellular toxins, thereby enabling fine-tuned regulation of plant physiological responses under stress [40].

To maintain RCS homeostasis, plants have evolved coordinated detoxification mechanisms involving both enzymatic and non-enzymatic systems. These include antioxidants and scavengers, amino acid-derived osmolytes, aldehyde dehydrogenases, aldo–keto reductases, and 2-alkenal reductases, which collectively limit excessive RCS accumulation and prevent irreversible cellular damage. Genetic and biochemical evidence further supports the role of RCS as key downstream mediators of ROS, linking oxidative stress perception to adaptive signaling as well as cellular toxicity [14,47,74].

A wide range of RCS are rapidly generated in plants under various abiotic stresses such as salinity, drought, heat, chilling, oxidative stress, and hormone-induced signaling [14,20,74]. Recent studies have emphasized the role of RCS as signaling molecules in plants exposed to diverse environmental stresses (Table 1). Under salinity and oxidative stress, lipid-derived RCS accumulate in Arabidopsis thaliana and halophytic species, where they modify protein thiol groups through Michael addition and Schiff base formation. At low concentrations, these reversible modifications regulate antioxidant capacity, ion homeostasis, and stress signaling pathways, whereas excessive RCS accumulation results in irreversible protein damage, growth inhibition, and metabolic dysfunction [15,20].

RCS have also been shown to induce PCD in the presence of H₂O₂ or NaCl in Arabidopsis and tobacco. In tobacco BY-2 cells, H₂O₂ treatment triggers the rapid production of acrolein and HNE, which in turn modulate intracellular ROS levels and fine-tune the progression of PCD, highlighting the dual role of RCS as both inducers and regulators of cell death [22]. In addition, RCS accumulation has been associated with aluminum stress responses, as elevated levels of multiple RCS have been detected in tobacco root tips under aluminum exposure [75].

Table 1. Abiotic stress-induced RCS generation and their signaling role in plants.

Stresses	Plant Species	RCS Generated	Targets (Protein/Lipid)	Effects/Functional Roles	References
Salinity	Arabidopsis thaliana	Acrolein, HNE, HHE	Reactive carbonyl species from PUFA oxidation in chloroplasts and mitochondria, and these RCS modify protein thiols for redox signaling at low levels but cause irreversible protein damage at high levels.	Lipid-derived RCS activate antioxidant defenses and stress tolerance in plants, while being detoxified by enzymes such as aldo-keto reductases.	[20]
	Eutrema parvulum	Acrolein, HNE, HHE	RCS mainly target membrane PUFAs to form toxic aldehydes and modify key antioxidant proteins via Michael addition, Schiff base formation, and cysteine oxidation.	Exhibited toxicity at high levels but act as stress signals at low levels, activating antioxidant defense, ROS signaling, and ion homeostasis, especially in halophytes.	[20]
	Oryza sativa	MDA	Membrane lipids, cellular proteins.	Inhibited germination, seedling growth, and biomass.	[76]
Drought	Arabidopsis thaliana	MDA, (E)-2-hexenal and (E)-2-butenal	PUFA-derived lipid peroxidation products (RSLVs, oxylipins, MDA) target membrane lipids and oxidatively modify proteins, including photosystem II components.	Activated stress-responsive genes through HSFA1-dependent and independent pathways to enhance heat tolerance.	[77]
Heat	Triticum aestivum	MDA, acrolein	Heat-shock proteins, photosystem II proteins (OEC33, PSII core proteins).	Protein aggregation, altered redox signaling, inactivation of oxygen-evolving complex, reduced photosynthesis.	[14]
	Oryza sativa	MDA, HNE, HHE	Lipid membranes, chloroplast proteins.	Membrane destabilization, reduced photosynthesis, oxidative damage, decreased photosynthetic efficiency.	[14]
	Arabidopsis thaliana	MDA, acrolein, HNE and HHE	Pollen proteins, reproductive enzymes.	Protein carbonylation, reduced pollen viability, impaired fertilization, reduced seed set and cell death.	[78]
Drought and heat	Arabidopsis thaliana	MDA	Thylakoid/PSII proteins (D1), membrane lipids, TFs and redox proteins.	Stress-combination-specific gene expression, dominant stomatal closure, impaired PSII repair, and activation of key stress TF families.	[54]
	Oryza sativa	MDA	Photosynthetic machinery, reproductive tissues (spikelets), reproductive sensitivity noted; lipid membranes.	Reprogrammed gene expression during spikelet development and combined drought-heat disrupts metabolism, seed filling, and yield.	[54]

Table 1. Abiotic stress-induced RCS generation and their signaling role in plants.

Stresses	Plant Species	RCS Generated	Targets (Protein/Lipid)	Effects/Functional Roles	References
Salinity	Arabidopsis thaliana	Acrolein, HNE, HHE	Reactive carbonyl species from PUFA oxidation in chloroplasts and mitochondria, and these RCS modify protein thiols for redox signaling at low levels but cause irreversible protein damage at high levels.	Lipid-derived RCS activate antioxidant defenses and stress tolerance in plants, while being detoxified by enzymes such as aldo-keto reductases.	[20]
	Eutrema parvulum	Acrolein, HNE, HHE	RCS mainly target membrane PUFAs to form toxic aldehydes and modify key antioxidant proteins via Michael addition, Schiff base formation, and cysteine oxidation.	Exhibited toxicity at high levels but act as stress signals at low levels, activating antioxidant defense, ROS signaling, and ion homeostasis, especially in halophytes.	[20]
	Oryza sativa	MDA	Membrane lipids, cellular proteins.	Inhibited germination, seedling growth, and biomass.	[76]
Drought	Arabidopsis thaliana	MDA, (E)-2-hexenal and (E)-2-butenal	PUFA-derived lipid peroxidation products (RSLVs, oxylipins, MDA) target membrane lipids and oxidatively modify proteins, including photosystem II components.	Activated stress-responsive genes through HSFA1-dependent and independent pathways to enhance heat tolerance.	[77]
Heat	Triticum aestivum	MDA, acrolein	Heat-shock proteins, photosystem II proteins (OEC33, PSII core proteins).	Protein aggregation, altered redox signaling, inactivation of oxygen-evolving complex, reduced photosynthesis.	[14]
	Oryza sativa	MDA, HNE, HHE	Lipid membranes, chloroplast proteins.	Membrane destabilization, reduced photosynthesis, oxidative damage, decreased photosynthetic efficiency.	[14]
	Arabidopsis thaliana	MDA, acrolein, HNE and HHE	Pollen proteins, reproductive enzymes.	Protein carbonylation, reduced pollen viability, impaired fertilization, reduced seed set and cell death.	[78]
Drought and heat	Arabidopsis thaliana	MDA	Thylakoid/PSII proteins (D1), membrane lipids, TFs and redox proteins.	Stress-combination-specific gene expression, dominant stomatal closure, impaired PSII repair, and activation of key stress TF families.	[54]
	Oryza sativa	MDA	Photosynthetic machinery, reproductive tissues (spikelets), reproductive sensitivity noted; lipid membranes.	Reprogrammed gene expression during spikelet development and combined drought-heat disrupts metabolism, seed filling, and yield.	[54]

Reports showed that exogenous applications of HNE, HHE, and acrolein at low concentration enhanced root growth and biomass accumulation in the halophytic model plant Eutrema parvulum under salt stress, potentially by stimulating H₂O₂-scavenging enzymes and improving ion homeostasis [17]. In guard cells, RCS such as acrolein and HNE act downstream of ROS and abscisic acid signaling to regulate Ca²⁺ influx, inhibit inward-rectifying K⁺ channels, and promote stomatal closure, thereby contributing to early stress adaptation and water-use efficiency [79,80]. Conversely, under combined drought and heat stress, excessive RCS-mediated protein carbonylation disrupts PSII repair, impairs reproductive development, and reduces yield in crops such as Arabidopsis and rice [54,78]. Nevertheless, plants mitigate RCS-induced cytotoxicity through efficient detoxification pathways, including aldo–keto reductases and glutathione-dependent conjugation systems, underscoring the central role of RCS as both damaging agents and critical signaling molecules in abiotic stress tolerance [14,74,81].

4. Crosstalk Between ROS and RCS in Stress Signaling

ROS and RCS form an integrated signaling module that enables plants to translate rapid redox changes into sustained stress responses (Figure 2), which operates in close coordination with other signaling networks, including sugar, hormonal, and metabolic pathways [82]. While ROS are well-established second messengers in plant stress signaling, their high reactivity and short lifetime necessitate the involvement of more stable downstream mediators. Oxidative modification of membrane polyunsaturated fatty acids by ROS generates lipid-derived RCS, including α,β-unsaturated aldehydes such as acrolein and 4-hydroxy-(E)-2-nonenal (HNE), which act as electrophilic signaling intermediates linking ROS production to downstream cellular responses [13,14]. Through covalent modification of thiol-containing proteins, RCS modulate ion transport, redox buffering, and kinase activity, thereby extending and fine-tuning ROS-dependent signaling cascades [47].

Guard cells provide a well-defined model for elucidating ROS-RCS crosstalk during stress and hormone signaling. Abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid induce stomatal closure. These hormonal signals lead to the generation of ROS. The generated ROS then promote lipid peroxidation, resulting in the accumulation of RCS. RCS act downstream of ROS and upstream of Ca²⁺-dependent signaling events in guard cells [18,79].

Consistent with this hierarchy, ABA-induced ROS production remains intact in cpk6 mutants, whereas stomatal closure triggered by acrolein or HNE is strongly impaired, identifying RCS as critical intermediates linking ROS to Ca²⁺-dependent protein kinase 6 (CPK6) activation [18]. MeJA signaling similarly requires ROS-dependent RCS production and selectively engages CPK6, but not CPK3, highlighting isoform-specific decoding of RCS signals in jasmonate responses [83,84]. This hierarchical organization with ROS as fast primary signals and RCS as slower but more stable secondary signals may enable temporal integration and amplification of stress inputs, although this hypothesis requires quantitative testing through mathematical modeling and kinetic measurements.

Beyond protein kinases, metabolic enzymes contribute to RCS signal transduction in guard cells. Myrosinases TGG1 and TGG2, which are highly expressed in Arabidopsis guard cells, function redundantly downstream of RCS accumulation and upstream of Ca²⁺ signaling. Loss of both TGG1 and TGG2 abolishes acrolein-induced stomatal closure, cytosolic alkalization, and ROS-associated Ca²⁺ elevation, whereas single mutants retain these responses, indicating that myrosinases are essential components of RCS-mediated signaling pathways [80]. These findings collectively support a conserved ROS → RCS → Ca²⁺ signaling axis with stimulus-specific downstream modulation.

RCS signaling is further integrated with glutathione (GSH)-dependent redox regulation. Electrophilic RCS readily conjugate with GSH, leading to transient GSH depletion and sensitization of guard cells to ROS, thereby reinforcing redox signaling loops without directly affecting ROS production itself [18,81]. This separation of ROS generation from downstream redox buffering underscores the role of RCS as modulators rather than mere by-products of oxidative stress.

Importantly, emerging evidence indicates that ROS-induced RCS formation is not restricted to guard cells. In rice, salt stress-induced RCS accumulation negatively affects germination and early seedling growth, and enhanced RCS detoxification capacity is closely associated with stress tolerance, extending the physiological relevance of ROS–RCS crosstalk from cellular signaling to whole-plant stress adaptation [76].

Beyond these cellular and tissue-level processes, ROS-RCS interactions also operate at the organellar scale to coordinate intracellular communication pathways. In particular, chloroplast-derived redox signals play a central role in mediating retrograde signaling to the nucleus under stress conditions [85,86]. In this context, plastid-derived ROS, particularly singlet oxygen (¹O₂) and hydrogen peroxide (H₂O₂), act as primary retrograde signals that integrate environmental stress cues with developmental programs, enabling dynamic plastid–nucleus coordination [87]. These ROS further promote lipid peroxidation processes, leading to the generation of RCS, including electrophilic oxylipins, which function as secondary messengers and can also directly regulate nuclear gene expression [88]. Notably, singlet oxygen-mediated signaling has been shown to control photosynthesis-associated nuclear genes via RCS-dependent pathways, highlighting a tightly coupled ROS–RCS signaling module [88]. Collectively, this ROS–RCS cascade constitutes a hierarchical, two-tier regulatory system that fine-tunes nuclear gene expression and facilitates rapid acclimation and stress adaptation in plants.

5. Research Gaps and Future Perspectives

Despite substantial progress in elucidating the roles of ROS and RCS as central regulators of plant stress acclimation, significant conceptual and challenges in translating laboratory findings to field applications remain. ROS and RCS signaling is highly context-dependent, governed by species identity, concentration thresholds, subcellular origin, and temporal dynamics. Although chloroplasts, mitochondria, peroxisomes, and the apoplast are recognized as major production sites [89], quantitative information on the local abundance and spatiotemporal behavior of specific ROS and RCS, such as singlet oxygen, hydrogen peroxide, and lipid-derived aldehydes, within discrete signaling microdomains remains limited. This lack of resolution hampers clear discrimination between signaling-relevant redox events and nonspecific oxidative damage. In addition, accurate quantification of cellular RCS levels remains technically challenging due to their high reactivity, low steady-state concentrations, and rapid metabolism. Most studies employ derivatization-based methods (e.g., 2,4-dinitrophenylhydrazine for carbonyl detection) or antibodies against RCS–protein adducts (e.g., anti-HNE-Michael adduct antibodies). However, these approaches provide limited spatiotemporal resolution and cannot distinguish free RCS from protein-bound forms. Development of genetically encoded RCS sensors analogous to roGFP-based ROS sensors represents a key technical frontier for the field.

Key gaps also persist in understanding the mechanisms of inter-organellar and long-distance ROS-RCS signal transmission. While organelle-to-nucleus communication is essential for stress-responsive gene regulation [90], the molecular intermediates that mediate these processes, and the integration of RCS within broader redox networks involving reactive nitrogen and sulfur species, remain poorly defined. At the molecular level, the spectrum of RCS-responsive targets and the functional consequences of their post-translational modification are incompletely characterized in plants, limiting insight into how RCS signals are decoded and coordinated with ROS production, calcium signaling, and phytohormone pathways.

From an applied perspective, ROS-RCS-mediated tolerance mechanisms often fail to translate from controlled environments to the field, particularly under combined stress conditions. Future progress will require high-resolution, integrative approaches, such as genetically encoded redox sensors, single-cell imaging, spatial omics, and redox proteomics, combined with systems biology and field-based validation. A deeper understanding of ROS-RCS crosstalk and its integration within complex redox signaling networks will be critical for translating redox biology into robust strategies for developing climate-resilient crops.