antioxidants

Review
Reactive Oxygen Species in Plants: From Source to Sink
Sheikh Mansoor 1 , Owais Ali Wani 2 , Jafar K. Lone 3 , Sweeta Manhas 1 , Navneet Kour 1 , Pravej Alam 4 ,
Ajaz Ahmad 5 and Parvaiz Ahmad 6, *

1 Division of Biochemistry, FBSc, SKUAST-J, Jammu 193201, Jammu and Kashmir, India;
[email protected] (S.M.); [email protected] (S.M.); [email protected] (N.K.)
2 Division of Soil Sciences and Agricultural Chemistry, SKUAST Kashmir,
Jammu 193201, Jammu and Kashmir, India; [email protected]
3 ICAR-NBPGR, Division of Germplasm Evaluation Pusa Campus, New Delhi 110012, India;
[email protected]
4 Department of Biology, College of Science and Humanities, Prince Sattam Bin Abdulaziz University,
Alkharj 11942, Saudi Arabia; [email protected]
5 Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia;
[email protected]
6 Department of Botany, GDC, Pulwama 192301, Jammu and Kashmir, India
* Correspondence: [email protected]

Abstract: Reactive oxygen species (ROS, partial reduction or derivatives of free radicals) are highly
reactive, dangerous and can cause oxidative cell death. In addition to their role as toxic by-products
of aerobic metabolism, ROS play a role in the control and regulation of biological processes such as
growth, the cell cycle, programmed cell death, hormone signaling, biotic and abiotic stress reactions
and development. ROS always arise in plants as a by-product of several metabolic processes that



are located in different cell compartments, or as a result of the inevitable escape of electrons to


oxygen from the electron transport activities of chloroplasts, mitochondria and plasma membranes.
Citation: Mansoor, S.; Ali Wani, O.;
These reactive species are formed in chloroplasts, mitochondria, plasma membranes, peroxisomes,
Lone, J.K.; Manhas, S.; Kour, N.;
Alam, P.; Ahmad, A.; Ahmad, P.
apoplasts, the endoplasmic reticulum and cell walls. The action of many non-enzymatic and enzy-
Reactive Oxygen Species in Plants: matic antioxidants present in tissues is required for efficient scavenging of ROS generated during
From Source to Sink. Antioxidants various environmental stressors. The current review provides an in-depth look at the fate of ROS
2022, 11, 225. https://doi.org/ in plants, a beneficial role in managing stress and other irregularities. The production sites are also
10.3390/antiox11020225 explained with their negative effects. In addition, the biochemical properties and sources of ROS
generation, capture systems, the influence of ROS on cell biochemistry and the crosstalk of ROS with
Academic Editors: Mohsin Tanveer,
Zhong-Hua Chen, Yizhou Wang and
other signaling molecules/pathways are discussed.
Ricardo Aroca
Keywords: reactive oxygen species; production; stress; signaling; cell death
Received: 5 December 2021
Accepted: 16 January 2022
Published: 25 January 2022

Publisher’s Note: MDPI stays neutral 1. Introduction

with regard to jurisdictional claims in Oxygen-evolving photosynthetic organisms introduced O2 to the reducing atmosphere
published maps and institutional affil- of Earth about 2.7 million years ago. This was further accompanied by the production of
iations. reactive oxygen species (ROS) as by-products of various metabolic reactions. In plants, dif-
ferent cellular organelles such as chloroplasts, peroxisomes and mitochondria are confined
for the generation of ROS. In addition to the above-mentioned sites of ROS production,
cell walls and plasma membranes containing peroxidases and amine oxidases, respectively,
Copyright: © 2022 by the authors.
lead to ROS production in case of environmental stress. ROS include singlet oxygen (1 O2 ),
Licensee MDPI, Basel, Switzerland.
This article is an open access article
hydrogen peroxide (H2 O2 ), superoxide (O2− ) and hydroxyl radicals (OH. ). They exhibit
distributed under the terms and
a variety of physiological responses, structural changes and the degradation of macro-
conditions of the Creative Commons molecules [1]. In the process of photosynthetic electron transport, chloroplasts consistently
Attribution (CC BY) license (https:// produce oxygen, which becomes eliminated by reduction and assimilation. In photosystem
creativecommons.org/licenses/by/ I (PS I) and the photorespiratory cycle, the photoreduction of O2 to a superoxide radical
4.0/). takes place by reduced components of the electron transport system. In the case of stress

Antioxidants 2022, 11, 225. https://doi.org/10.3390/antiox11020225 https://www.mdpi.com/journal/antioxidants

Antioxidants 2022, 11, 225 2 of 14

and the low availability of CO2 , the enzyme RUBP (ribulose-1,5-bisphosphate) carboxy-
lase/oxygenase prefers oxygen as a substrate to catalyze a reaction. It leads to glycolate
production, which on oxidation, produces H2 O2 in peroxisomes via glycolate oxidase. In
the case of the plasma membrane, multimeric cytochromes present in the electron trans-
port chain favor the reduction of oxygen to superoxide (O2 − ) via NADPH-dependent
oxidases. Unlike the plasma membrane, pH-dependent peroxidase, oxalate oxidase and
amine oxidase are responsible for ROS generation in the apoplast [2–4].
In mammals, mitochondria are major sites for ROS production, whereas a less-
prominent role has been observed in plants [5–8]. Many studies assume that ROS pro-
duction is a basic symptom of plant toxicity under abiotic stress. As the production of
ROS increases, the phytotoxicity also rises. In such toxic conditions, plant growth and
metabolism are adversely affected, thereby leading to less crop productivity. Despite the
fact that ROS are involved in the cellular metabolism of plant cells, their overproduction
under stress conditions causes photooxidative damage to various cellular entities such
as carbohydrates, lipids, nucleic acid and proteins [9]. In addition to ROS generation,
their regulation is mainly achieved by defensive mechanisms, including enzymatic as well
as non-enzymatic antioxidants. In the enzymatic system, superoxide is responsible for
the conversion of hydroxyl to hydrogen peroxide, which is catalyzed by peroxidase and
catalase (CAT) to produce water and dioxygen. The non-enzymatic scavenging mecha-
nism is mediated by low-molecular-mass antioxidants, namely glutathione, ascorbic acid,
carotenoids, flavonoids, etc., by targeting hydroxyl radicals and singlet oxygen. In case
of an imbalance between the production and scavenging of ROS, plant cells generate ox-
idative stress, eventually leading to oxidative modification and ultimately cell death [10].
As a secondary messenger, ROS take part in cell division, differentiation, biotic and abi-
otic responses as the cells are maintained in a reduced state due to low ROS levels [11].
Therefore, ROS play a crucial role, and maintaining a threshold level is important as it is
necessary for plant growth and development. Apart from the damaging effect, ROS are
also considered to be a signaling molecule playing an immense role in plant growth and
development, stress-responsive genes and leading to programmed cell death. Through
various redox reactions, the signal is transported to the nucleus, which goes downstream
via the mitogen-activated protein kinase (MAPK) pathway. It helps in the development of
enhanced tolerance in several cellular mechanisms under stress conditions [12,13]. Many
studies demonstrated the critical role of H2 O2 in several crop plants, such as rice, maize,
wheat, bean and soybean, in the regulation of a stress-responsive environment. Apart from
ROS, reactive nitrogen species, reactive sulfur species and reactive carbonyl species exhibit
roles in signaling as well as in the stress tolerance mechanism [8].
Various stress factors, including drought, salinity, temperature, heavy metal, etc.,
are known for disturbing the equilibrium maintained between ROS generation and its
scavenging. In such conditions, the capability of plant tolerance mainly relies upon some
prime factors: extremity and time duration of stress; variation in growth; how much faster
the plant is likely to adapt with respect to changes in the environment [14]. At times,
manifold stressors are reported to occur simultaneously, which thereby leads to a rapid
decline in crop productivity [2,14]. To keep up with drastic environmental conditions, plants
have evolved various stress-responsive genes encoding their respective proteins required
for activation as well as regulation of ROS. Herein, among several genes, transcription
factors play a prominent role in providing increased tolerance, and many studies have
demonstrated their role in the successful management of abiotic stress [15–18]. In this
review, we provide detailed insight regarding ROS generation, their biochemistry and
the signaling mechanism in plants to effectively cope with abiotic stress. In particular,
we summarize strategies/plant disease resistance mechanisms, the antioxidant defense
systems and also the crosstalk of ROS with the signaling molecules.
Antioxidants 2022, 11, 225 3 of 14

2. ROS in Disease Resistance

The adaptability of plants towards pathogen attack and strategies of co-evolution have
always been incremental and of prime importance for agricultural production systems
over the years. Plant susceptibility and resistance against foreign invasion by pathogens
are under the control of recognition and signaling pathways between the host and the
pathogen. Microbe/pathogen-associated molecular patterns, i.e., MAMPs/PAMPs and
Avr (avirulent) gene products that correspond to their host receptors (plants) along with
R (resistance) genes, lead to the activation of a signaling cascade that generates ROS,
phytoalexins and anti-microbial genes. This further activates a wide range of plant defense
genes proficient against a wide range of pathogens. In plants, the electron transport chain in
mitochondria and chloroplasts, as well as peroxisomal photorespiration, generate ROS [19].
RBOH (respiratory burst oxidase homolog) genes encoding NADPH oxidase, polyamine
oxidase (PAO)-mediated degradation of spermidine and oxalate oxidase are involved in
the production of apoplastic ROS in the plasma membrane.
The most stable and abundant ROS in plants include H2 O2 (hydrogen peroxide), su-
peroxide (O2 − ), (hydroxyl) (OH) and singlet oxygen (1 O2 ). There is a rapid interconversion
between the four that provides a higher functional variability. Of all the four classes, H2 O2
is highly stable and ROS-transported via aquaporin membranes [20]. Different concentra-
tions of ROS generated have different physiological outcomes. In small concentrations,
signaling functions are exerted by ROS. However, the extensive accumulation of ROS may
lead to cell death caused by its damaging oxidative effects on nucleic acids, proteins and
lipids. An interplay between calcium channels, NADPH oxidases (NOX) and calcium
fluxes induced during oxidative stress generates a ROS wave, which can transduce long-
distance signals [21]. An ROS wave is a respiratory burst-homolog-D (RBOHD)-mediated
cell–cell self-propagating process of ROS production. Once triggered, it results in enhanced
production of ROS in a single cell, which acts as a sensory signal for other cells to increase
their ROS production. In a recently reported novel function of the ROS wave, there is
coordination between different induced stresses and the whole-plant systemic stomatal
response [21–23]. The ROS wave is a cell-to-cell auto-propagating process of ROS produc-
tion mediated by the respiratory burst homolog D (RBOHD) protein [4,14,21,24]. Once
triggered in a single cell, it causes the enhanced production of ROS by the cell. This results
in the accumulation of ROS at the apoplast (RBOHD produces ROS at the apoplast) [25].

3. Regulation of MAP Kinase

Among the mitogen-activated protein kinases (MAPKs), MAP2Ks and MAP3Ks repre-
sent a class of key signal transduction proteins that are triggered in response to a plethora
of developmental and environmental factors. ROS accumulation is well-known to be the
activating factor of MAPK signaling under biotic and abiotic stress conditions. MAPK
pathways are three-rung kinases of MAPKKK that phosphorylate and activate MAPKK,
which in turn activate MAPKs after phosphorylation. The phosphorylation of MAPKs,
once activated, leads to the inactivation or activation of various target proteins as well
as transcription factors. MAPKS are involved in pathogen-mediated signal transduction
(biotic) and abiotic stress such as drought, cold, salinity, wounding, ROS, ozone and hor-
mone stimuli [26,27]. Although ROS production inevitably mediates stress-induced defense
responses, it is equally important to limit over-accumulation of ROS. To prevent oxidative
stress induced by ROS overproduction, the cell responds to the increased production of
ROS by an antioxidant defense system, which has a key role in maintaining the levels
of ROS. Thus, redox control of TFs (transcription factors) is critical in defining the cellu-
lar response to oxidative stress and gene expression profiling (Figure 1). Several genetic
studies have reported the significance of antioxidant enzymes in maintaining a positive
correlation between plant stress tolerance and the expression of enzymes responsible for
ROS regulation. The scavenging of ROS is accomplished via non-enzymatic as well as
enzymatic pathways. Low-molecular-weight metabolites glutathione, alpha-tocopherol,
ascorbate, flavonoids and carotenoids mediate the non-enzymatic antioxidant pathway.
oxidants 2022, 11, x FOR PEER REVIEW 4 of 14

Antioxidants 2022, 11, 225 4 of 14

enzymatic pathways. Low-molecular-weight metabolites glutathione, alpha-tocopherol,
ascorbate, flavonoids and carotenoids mediate the non-enzymatic antioxidant pathway.

Figure 1. ROS signaling, the activation of the MAPK cascade and redox ROS homeostasis in the cell.
Figure 1. ROS signaling, the activation of the MAPK cascade and redox ROS homeostasis in the
In biotic and abiotic stressors, the reactive oxygen species (ROS) signaling pathway is regulated by
cell. In
mitogen-activated biotic and
protein abiotic
kinases stressors,
(MAPK). ROS theisreactive oxygen
a common species (ROS)
messenger that issignaling
producedpathway
in re- is regulated
by mitogen-activated protein kinases (MAPK). ROS is a common messenger
sponse to both the stress response and the MAPK cascade. Despite having a similar MAPK signaling that is produced in
response to both the stress response and the
regulator, the plant's reaction to both stressors is distinct. MAPK cascade. Despite having a similar MAPK signaling
regulator, the plant’s reaction to both stressors is distinct.
The major enzyme classes involved in the enzymatic regulation of ROS include ascor-
bate peroxidases, Thesuperoxide
major enzyme classes involved
dismutases, in theglutathione
catalases, enzymatic regulation of ROS
reductases, dehy-include ascor-
bate peroxidases, superoxide dismutases, catalases, glutathione
droascorbate reductases and monodehydroascorbate reductases. Thio-, gluta- and perox-reductases, dehydroascor-
bate reductases
iredoxins, along and monodehydroascorbate
with peroxidases reductases.
and glutathione peroxidases, are Thio-,
potentgluta- and peroxiredoxins,
ROS scaven-
gers. Functional and sequence analyses of the Arabidopsis genome revealed 80 MAP- Functional
along with peroxidases and glutathione peroxidases, are potent ROS scavengers.
and sequence
KKKs, 10 MAPKKs and 20 analyses
MAPKs, of the Arabidopsis
presenting a similargenome revealed
gene catalog 80 MAPKKKs,
to those observed in10 MAPKKs
other plant genomes. Along with MAPK proteins, plant hormones have a keyin
and 20 MAPKs, presenting a similar gene catalog to those observed other
role plant genomes.
in ac-
Along with MAPK proteins, plant hormones have a key role in acclimation responses to
climation responses to abiotic stress. Recent studies have elucidated an interplay between
abiotic stress. Recent studies have elucidated an interplay between ROS and phytohor-
ROS and phytohormones during abiotic stress. An increase in ROS levels under abiotic
mones during abiotic stress. An increase in ROS levels under abiotic stress not only affects
stress not only affects the transcription of genes, metabolic flux and proteome, but also
the transcription of genes, metabolic flux and proteome, but also modulates the function
modulates the function and level of phytohormones [28]. A molecular link between oxi-
and level of phytohormones [28]. A molecular link between oxidative stress and the auxin
dative stress and the auxin signal transduction pathway has long been reported [29–34].
signal transduction pathway has long been reported [29–34]. The production of ROS during
The production of ROS during stress (abiotic) results in changes in auxin gradients that,
stress (abiotic) results in changes in auxin gradients that, in turn, reduce auxin-induced
in turn, reduce auxin-induced signaling. Auxins can also induce ROS production and help
signaling. Auxins can also induce ROS production and help in maintaining ROS homeosta-
sis, exhibiting an association between oxidative stress and auxin signaling [35]. Auxins
also play a role in the activation of RAC/ROP (Rho-GTPase), which further interact with
Antioxidants 2022, 11, 225 5 of 14

NADPH oxidases that cause apoplastic production of ROS. On the contrary, ROS regulate
the transport of auxins via PIN gene regulation and the relocation of auxin exporters. Auxin
signaling is followed by a mitogen-activated protein kinase (MAPK) cascade, resulting in
crosstalk between auxins, MAPKs and ROS. It has been reported that MPK12, an Arabidop-
sis protein, interacts with IBR5 (indole-3-butyric acid response), a MAPK phosphatases
specifically, which on dephosphorylation inactivates MPK12. Thus, MPK12 acts as an IBR5
phosphatases substrate that is activated by auxins in vivo, and the suppression of MPK12
results in auxin-responsive gene expression, suggesting the role of MPK12 as a negative
regulator in Arabidopsis auxin signaling [8].

4. ROS Mediated Stress Responses—Stress, Hormone and ROS Crosstalk

ROS is a commonly produced factor under both abiotic as well as biotic stress. In Ara-
bidopsis, mitogen-activated protein kinase kinase kinase 1 (MEKK1), MAP3K is activated
under abiotic stress conditions such as wounds, cold, salt and drought, and in biotic stress
responses against fungal and bacterial elicitors, MEKK1 is known to be activated with the
production of ROS in such stimuli. When there are abiotic stimuli, the MKK2-MPK4/6
module is activated by MEKK (mitogen-activated protein kinase kinase 2) [36]. On the other
hand, biotic stimuli result in the activation of the MKK4/5-MPK3/6-VIP1/ACS6 module.
MEKK1-MKK1/2-MPK4 acting upstream of MKS1/WRKY33 also works in mediating
pathogen-related responses. ROS, when produced under different kinds of environmental
stresses, such as heavy metal, ozone, abscisic acid (ABA) treatment and biotic stress, ac-
tivates MPK6 and MP3K, which further mediate different responses [36,37]. MPK6 and
MP3K act downstream of Ser/Thr protein kinase OXI1 in Arabidopsis, which has two
different biological roles; it stimulates fungal pathogen resistance in plants and stimulates
root development. In rice, hydrogen peroxide activates MPK6 and MPK3 kinases, which
are involved in resistance against abiotic stresses such as salt, UV rays, cold and heavy
metal, as well as biotic stress resistance against fungal pathogen. What makes a single
pathway act in two processes can be explained from the above-cited examples where
crosstalk mediated by ROS among MAPKs suggest that in different environmental factors,
the production of ROS results in the activation of MAPKs (similar), but their interaction
and final response towards different stresses becomes fundamentally different. It seems
that ROS acts as a messenger that is involved in encoding information for the activation of
different responses [37]. The complexity of the intricate interplay between ROS and the
plant hormones further increases under multiple stress conditions [38]. For instance, inter-
actions between ROS, ABA, jasmonic acid (JA) and salicylic acid (SA) are associated with
the regulation of stomatal movement during stress combination. It has been reported that
abil (ABA-insensitive mutants), having impaired ABA function, and ABII (ROS regulated
protein), were sensitive towards the combined stress of drought and high temperatures.
The mutants were also sensitive to combined high temperature and salinity stresses, thus
highlighting the role of ROS–ABA interactions in the acclimation of plants to combination
stress. Following a combination of high temperatures and drought stress, the stomatal
closure was accompanied by an increase in hydrogen peroxide and JA concentration with a
decrease in SA concentration in the leaves, which suggests the possibility of a canonical
role of H2 O2 and JA in stomatal responses independent of ABA [39–41].

5. Defense System against ROS Production and Accumulation

Introduction to the Defense System against ROS Production and Accumulation
The ROS defense system in plants and its various processes and components were
discovered at the end of the twentieth century [34,42–45]. Recently, it was concluded
that the defense system against ROS is not only due to the scavenging system, but it also
constitutes an enzymatic as well as non-enzymatic defense system, and these defense
systems are initiated by various external environmental stresses [46]. Routine cellular
metabolism results in the production of ROS, which is regulated by various enzymatic and
non-enzymatic defense systems (Table 1). Enzymatic defense systems include APX, CAT,
 the defense system against ROS is not only due to the scavenging system, but it also con-
stitutes an enzymatic as well as non-enzymatic defense system, and these defense systems
Antioxidants 2022, 11, 225 are initiated by various external environmental stresses [46]. Routine cellular metabolism 6 of 14
results in the production of ROS, which is regulated by various enzymatic and non-enzy-
matic defense systems (Table 1). Enzymatic defense systems include APX, CAT, SOD and
GPX,
SOD andwhereas
GPX, whereasnon-enzymatic systemssystems
non-enzymatic includeinclude
glutathione (GSH),(GSH),
glutathione ascorbic acid acid
ascorbic (AA),
phenolic
(AA), compounds
phenolic compounds andandtocopherols
tocopherols(TOCs)(TOCs)[45,47,48].
[45,47,48]. Plants possess aamultifarious
Plants possess multifarious
ROSdefense
ROS defensesystem
systemcomprised
comprisedofofboth bothenzymatic
enzymaticasaswellwellasasnon-enzymatic
non-enzymaticsystem. system.ROSROS
production,asaswell
production, wellasasscavenging,
scavenging,can canbebelocated
locatedinindifferent
differentcell
cellcomponents,
components,such suchasas
peroxisomes,chloroplasts
peroxisomes, chloroplastsand andmitochondria,
mitochondria,and andthere
thereisisstrong
strongcoordination
coordinationbetween
between
theseorganelles
these organellesin in the
the case of of such
suchpathways
pathways[49].[49].Under
Undernormal
normal conditions,
conditions,there is equi-
there is
equilibrium between
librium between thethe productionand
production andscavenging
scavengingof of ROS
ROS in plants,
plants, but
butunder
understress,
stress,this
this
equilibrium
equilibrium is is
disturbed,
disturbed, leading
leading to an elevation
to an in ROS
elevation levels
in ROS [49],[49],
levels which leads
which to oxidative
leads to oxida-
stress to cell to
tive stress components, whereas
cell components, in moreinevolved
whereas plants, there
more evolved plants,is athere
natural
is adefense
naturalsystem
defense
tosystem
countertothis rise inthis
counter ROS levels
rise in ROS[50].levels
The ROS
[50].defense
The ROS system
defenseis very important
system is veryto reduce
important
ROS levels in
to reduce ROS plants
levelsduring abiotic
in plants stresses.
during abioticWith time, With
stresses. plantstime,
haveplants
evolvedhave complex
evolved
defense systems against the accumulation and production of ROS (Figure
complex defense systems against the accumulation and production of ROS (Figure 2) [51]. 2) [51].

Figure2. 2.Effects
Figure Effectsofofoxidative
oxidativestresses
stressesononplant
plantparts
partsand
anddifferent
different defensemechanisms.
defense mechanisms.InInplants,
plants,
ROS cause serious damage to the cells by inhibiting proteins, DNA and other metabolic pathways.
ROS cause serious damage to the cells by inhibiting proteins, DNA and other metabolic pathways.
Conversely, the defense system is activated in the plants against ROS to regulate its functional ac-
Conversely, the defense system is activated in the plants against ROS to regulate its functional activity
tivity by activating different enzymatic and non-enzymatic antioxidant agents.
by activating different enzymatic and non-enzymatic antioxidant agents.

6.6.Enzymatic
EnzymaticDefense
DefenseSystems
Systems
Enzymatic
Enzymatic defense
defense systems
systems against
against ROS ROS include
include various
various enzymes
enzymes suchsuch as glutathi-
as glutathione
one reductases (GRs), dehydroascorbate reductases (DHARs),
reductases (GRs), dehydroascorbate reductases (DHARs), superoxide dismutases superoxide dismutases
(SODs),
(SODs), monodehydroascorbate reductases (MDHARs), glutathione
monodehydroascorbate reductases (MDHARs), glutathione peroxidases (GPXs), catalases peroxidases (GPXs),
catalases (CATs) and ascorbate peroxidases (APXs). The stresses
(CATs) and ascorbate peroxidases (APXs). The stresses associated with different defenseassociated with different
defense mechanisms
mechanisms are listedarein listed
Table in
1. Table
Various1. Various
enzymes enzymes
involved involved
in the in the defense
defense system system
are
are discussed,
discussed, such such as SOD
as SOD from from the metallo-enzyme
the metallo-enzyme family family of enzymes.
of enzymes. The mechanism
The mechanism by
by which
which they they decrease
decrease ROS includes
ROS includes the removal of O2 −of, converting
the removal O2−, converting it intoit O
into
2 O
and 2Hand H
2 2 by
O 2O2

a by a dismutation
dismutation process
process that reduces
that reduces the formation of OHof− OH
the formation [52].− [52]. In plants,
In plants, SODsSODs are pre-
are present
insent in chloroplasts
chloroplasts (Cu/Zn-SOD),
(Cu/Zn-SOD), peroxisomes
peroxisomes (Cu/Zn-SOD)
(Cu/Zn-SOD) and mitochondria
and mitochondria (Mn-SOD). (Mn-
SOD). SOD in plants is increased by both biotic as well as abiotic stresses
SOD in plants is increased by both biotic as well as abiotic stresses [52,53]. CAT are heme- [52,53]. CAT are
heme-containing
containing enzymesenzymes that two
that degrade degrade two molecules
molecules of H
of H2 O2 into H2O2O 2 into
and OH22O byand O2 by a dis-
a dismutation
mutation
process. process.
Their tissueTheir tissue selectiveness
selectiveness depends ondependsthe place onwhere
the placetheywhere they are
are present present
[54]. The
expression of CAT is usually increased by drought, cold, heat and UV radiation [55,56].
Ascorbate-glutathione (AsA-GSH) has various reactions that enable the disposal of H2 O2 .
This enzyme is mainly functional in chloroplasts, cytosol and mitochondria [57,58]. Plant
glutathione-S-transferases (GSTs) are involved in programmed cell death and the response
Antioxidants 2022, 11, 225 7 of 14

to biotic and abiotic stresses [59,60]. The enzymatic defense system against ROS is highly
evolved as per the type of stress and place where there is oxidative damage.

Non-Enzymatic Defense Systems

Plants are armed with non-enzymatic and low molecular antioxidants such as carotenoids,
α-tocopherol, GSH, AsA and proline. These are also involved in retrograde signaling [61,62].
GSH and AsA are the main elements that reduce H2 O2 as they are quickly regenerated.
The GSH-AsA cycle is responsible for the removal of ROS [63]. AsA acts as a cofactor as
well as being responsible for the formation of tocopherol. GSH also plays an important
role in ROS scavenging and as a raw material for various peroxidases. GSH can be located
in mitochondria, nuclei, the endoplasmic reticulum and vacuoles [64]. Tolerance to high
temperatures, drought and salinity is also enabled by the GSH content in plants [65,66].
Tocopherols are fat-hating compounds responsible for the disintegration of ROS; hence,
they also protect membranes [67]. Carotenoids are lipid-soluble and mostly present in
plastids. An enhanced concentration of these helps to build tolerance against various
abiotic stresses [68]. Blue, red and purple pigmentation is provided by flavonoids in seeds,
flowers and fruits. They are also reported to protect plants against various abiotic stresses
such as high temperature and UV radiation [69–72].

Table 1. Different types of plants and their associated stresses and defense mechanisms.

Plant Type of Stress Defense System Reference

Triticum aestivum Drought CAT and SOD activity increased [73]
Brassica napus Drought Increased POD and CAT activity [74]
Decreased ascorbate and increased DHA while
Vigna radiata Drought [75]
decrease in their ratio
Vigna radiata Salinity Enhanced ascorbate and DHA activity [76]
Orysa sativa L. Salinity Enhanced GSH and GB content, enhanced SOD activity [66]
Portulaca oleracea L. Elevated temperature Increased SOD and POD activity [77]
Gossypium hirsutum Elevated temperature Increased FeSOD and Cu/ZnSOD activity [78]
Triticum spp. Freezing temperature Increased GST and APX activity [79]
Camellia sinensis L. Freezing temperature Increased tea polyphenol to amino acid ratio [80]
Prunus persica L. Batsch Flooding Increased CAT, POD and SOD activity [81]
Glycine max L. Heavy metal Increased activity of both enzymes, i.e., SOD and POD [82]
Orysa sativa L. Heavy metal stress Decreased ascorbate and DHA [83]
S. lycopersicum L. High light SOD and POD activity decreased [52]
Malus crabapple High ozone Enhanced POD, CAT and SOD [74]
Medicago sativa L. Alkalinity stress Increased ascorbate, POD and CAT activities [80]
Triticum aestivum Acidic stress Decreased ascorbate and GSH activity [83]

7. Impact of ROS on Cell Biochemistry

Reactive oxygen species are produced in the form of partially reduced or excited
forms of atmospheric oxygen, e.g., singlet oxygen (1 O2 ), hydrogen peroxide (H2 O2 ) and
hydroxyl radical (OH. ) (Figure 3) [84]. They act as signaling molecules in cells but are also
thought to be unavoidable toxic products of aerobic metabolism [19,85–87]. In cell signaling,
the ROS molecules are highly versatile because of their diverse properties that include
sites of production, different levels of reactivity and crossing potential to the biological
membrane [1,61,88]. These molecules were first used by the cells to detect the harmful
levels of atmospheric oxygen, or to regulate different metabolic activities, but they have
evolved as potent signaling molecules to regulate almost all aspects of life in plants, animals
and other eukaryotic organisms [24]. In the plant kingdom, these molecules regulate cell
differentiation and development, stress signaling, plant interactions, systemic response,
 thought to be unavoidable toxic products of aerobic metabolism [19,85–87]. In cell signal-
ing, the ROS molecules are highly versatile because of their diverse properties that include
sites of production, different levels of reactivity and crossing potential to the biological
membrane [1,61,88]. These molecules were first used by the cells to detect the harmful
levels of atmospheric oxygen, or to regulate different metabolic activities, but they have
Antioxidants 2022, 11, 225 8 of 14
evolved as potent signaling molecules to regulate almost all aspects of life in plants, ani-
mals and other eukaryotic organisms [24]. In the plant kingdom, these molecules regulate
cell differentiation and development, stress signaling, plant interactions, systemic re-
cell death
sponse, celland
deathredox
and potentials [19,89–92].
redox potentials The process
[19,89–92]. of producing
The process the ROS
of producing molecules
the ROS mol-
occursoccurs
ecules eithereither
by aerobic metabolism
by aerobic or by or
metabolism cellular antioxidative
by cellular mechanisms
antioxidative constantly
mechanisms con-
occurring in cells to prevent oxidative damage/stress [84]. Therefore, many
stantly occurring in cells to prevent oxidative damage/stress [84]. Therefore, many antiox-antioxidant
systems
idant in thein
systems cell
thehelp
cellinhelp
maintaining the ROSthe
in maintaining level
ROSat level
a non-toxic level, and
at a non-toxic any deviation
level, and any
from this balance could generate ROS-signaling reactions.
deviation from this balance could generate ROS-signaling reactions.

Atmosphericoxygen
Figure3.3.Atmospheric
Figure oxygenisisshownshowntotoundergo
undergoexcitation
excitationororreduction
reductiontotoform
formdifferent
differentROS
ROS
andreactive
and reactivenitrogen
nitrogenspecies;
species;super
superoxide
oxidedismutase
dismutaseisisshown
shownto toform
formhydrogen
hydrogenperoxide
peroxide(H (H22OO2),2 ),
whichininturn
which turnreacts
reactswith
withFeFe2+2+totoform
formhydroxyl
hydroxylradicals
radicals(OH
(OH−)−via
) via
thethe Fenton
Fenton reaction.
reaction.

Thus,ROS
Thus, ROSsignaling
signaling is
is aa highly
highly regulated
regulatedprocess
processand andisismediated
mediatedbyby the accumulation
the accumula-
of ROS levels at specific cellular compartments. This may occur
tion of ROS levels at specific cellular compartments. This may occur by plasma-mem- by plasma-membrane-
bound NADPH-oxidase
brane-bound NADPH-oxidase in plants andand
in plants RBOH RBOH termed
termed as asNOX
NOXininanimals. Theseare
animals. These are
those enzymes that produce ROS in the apoplast [25,93–95]. This class of enzymes is alsois
those enzymes that produce ROS in the apoplast [25,93–95]. This class of enzymes
also found
found in otherin other cell organelles,
cell organelles, such such as endoplasmic
as endoplasmic reticulum
reticulum (ER), (ER), vacuoles,
vacuoles, nuclei,
nuclei, mi-
mitochondria or peroxisomes, and are mostly regulated via calcium
tochondria or peroxisomes, and are mostly regulated via calcium and other phosphoryla- and other phospho-
rylation/dephosphorylation
tion/dephosphorylation reactions
reactions (Figure (Figure 4) [25,94].
4) [25,94]. In addition,
In addition, ROSwere
ROS levels levelsmedi-
were
mediated by peroxisomes at the apoplast as well as in the chloroplast,
ated by peroxisomes at the apoplast as well as in the chloroplast, mitochondria and nuclei mitochondria and
nuclei [1,24,89]. Therefore, a balance occurs in ROS production between
[1,24,89]. Therefore, a balance occurs in ROS production between the metabolically gen- the metabolically
generated
erated levels,
levels, the ROS
the ROS diffusion
diffusion raterate
andand reactivity
reactivity andand
thethe removaland
removal andperception
perceptioninin
different cellular organelles, which on integration, generate a site-specific
different cellular organelles, which on integration, generate a site-specific signaling signaling system
sys-
to regulate the antioxidant process and overall determine the cell-specific response to the
tem to regulate the antioxidant process and overall determine the cell-specific response to
stimulus [24]. It is important to remember the action of ROS, with the course of evolution
the stimulus [24]. It is important to remember the action of ROS, with the course of evolu-
in aerobic organisms, requires a specific solution at each cellular organelle to regulate ROS
tion in aerobic organisms, requires a specific solution at each cellular organelle to regulate
at each stage of their lifecycle. Hence, depending upon the nature of beneficial and toxic
ROS at each stage of their lifecycle. Hence, depending upon the nature of beneficial and
roles, they are named under a specific terminology called the “double-edged sword of
toxic roles, they are named under a specific terminology called the “double-edged sword
life” [1,14,96,97]. Therefore, ROS are predominantly beneficial to cells, help in maintaining
cellular and biochemical processes and regulate oxidative stress and cell death such as
ferroptosis or necrosis [98,99].
 Antioxidants 2022, 11, x FOR PEER REVIEW 9 of 14

of life” [1,14,96,97]. Therefore, ROS are predominantly beneficial to cells, help in main-
Antioxidants 2022, 11, 225
taining cellular and biochemical processes and regulate oxidative stress and cell 9death
of 14

such as ferroptosis or necrosis [98,99].

Figure 4.4. Integration

Figure Integration ofof ROS
ROS and
andredox
redoxbiology
biologyinincellular
cellularprocesses.
processes.Different cellcell
Different organelles, in-
organelles,
cluding mitochondria, chloroplasts, peroxisomes and cell-wall-bound peroxidases
including mitochondria, chloroplasts, peroxisomes and cell-wall-bound peroxidases (PER) and (PER) and res-
piratory burst oxidase homologs (RBOHs), produce ROS that accumulate in the form of hydrogen
respiratory burst oxidase homologs (RBOHs), produce ROS that accumulate in the form of hydrogen
peroxide (H2O2), resulting in the mediation of cell-to-cell signaling pathways. However, the pres-
peroxide (H2+2 O2 ), resulting in the mediation of cell-to-cell signaling pathways. However, the presence
ence2+of Fe ions can cause cellular oxidative stress via hydroxyl radicals. These processes must be
of Fe ionsand
balanced canare
cause cellular
crucial for oxidative stress for
redox biology via the
hydroxyl radicals.
regulation These
of the processesand
metabolism mustother
be balanced
physio-
and are and
logical crucial for redox
cellular biology for the regulation of the metabolism and other physiological and
functions.
cellular functions.
8. Conclusions and Future Perspectives
8. Conclusions and Future Perspectives
The above discussion emphasizes ROS functioning as major determinants of cell fate
The above discussion emphasizes ROS functioning as major determinants of cell fate as
as well as pivotal drivers of plant growth and development. Phytohormones such as ab-
well as pivotal drivers of plant growth and development. Phytohormones such as abscisic
scisic acid and ethylene regulate stress-induced growth changes and control ROS produc-
acid and ethylene regulate stress-induced growth changes and control ROS production
tion through
through antagonistic/synergistic
antagonistic/synergistic interactions
interactions [32–34,80].
[32–34,80]. Components
Components of theof the
cell cell such
wall, wall,
such as the GASA/Snakin proteins, are also involved in the integration
as the GASA/Snakin proteins, are also involved in the integration of ROS signaling and of ROS signaling
and phytohormones,
phytohormones, thus controlling
thus controlling growth growth and development
and development processesprocesses
along withalong
pathogenwith
pathogen resistance in plants. Insights into the regulation of these
resistance in plants. Insights into the regulation of these proteins by apoplastic ROS andproteins by apoplastic
ROS functions
their and their will
functions
providewilla provide a better understanding
better understanding of the redox-dependent
of the redox-dependent integration in-
tegration of extracellular and intracellular signals involved in plant
of extracellular and intracellular signals involved in plant growth and development. At growth and develop-
ment. Atthere
present, present, there is currently
is currently a lack of aknowledge
lack of knowledge about stress-inducible
about stress-inducible effects oneffects
redoxon
redox post-translational
post-translational modifications
modifications that helpthat helpregulation
in the in the regulation
of proteinof functions
protein functions
and protein and
protein localization
localization [100]. The [100]. The knowledge
knowledge of PTMs is of essential
PTMs is toessential to completely
completely understand understand
the ROS-
the ROS-dependent
dependent mechanisms mechanisms
involved in involved in environment-related
environment-related plant growth plant
andgrowth and de-
development.
velopment. Research on redox-dependent programmed cell
Research on redox-dependent programmed cell death demonstrates that ROS levels maydeath demonstrates that ROS
levels may increase above two different threshold levels altogether
increase above two different threshold levels altogether with an increase in ROS production with an increase in
ROSexceeds
that production thatscavenging
cellular exceeds cellular scavenging
capacity, capacity,
both leading to cellboth leading
death to cell
[32]. The deathiden-
current [32].
The current
tification identification
of plant genes thatofareplant genes that
involved are involved inPCD
in ROS-dependent ROS-dependent
is solely the PCD is solely
beginning of
athe
newbeginning
era during of awhich
new era during which
excavating excavating
the surplus of genetheregulatory
surplus ofnetworks
gene regulatory
controlling net-
works
this controlling
mechanism this
will bemechanism
momentous. will be momentous.
Additionally, Additionally,
the screening the screening
of suppressors forofROS-
sup-
pressors for ROS-induced cell death and cloning the remaining alleles
induced cell death and cloning the remaining alleles has already been reported to regress has already been
reported
or to regress or induce
induce programmed cell deathprogrammed
in Arabidopsis cell death
mutants.in Arabidopsis
This will leadmutants. This will
to an increase in
lead
the to an increase
number of genesininvolved
the numberin the ofplant
genesPCD involved in theFinalized
pathways. plant PCD pathways. Finalized
transcriptome studies
on genomes have led to the identification of hundreds of genes rapidly induced by ROS
signaling [54]. Within this checklist, a group of novel genes involved in transduction and
early ROS perception will undeniably be located. Functional screening of up-regulation
Antioxidants 2022, 11, 225 10 of 14

or down-regulation of these genes and scoring them for their potential to regulate ROS-
dependent cell death will help in their identification. Using this approach, along with
the characterization of target proteins regulating post-translational modifications, such as
nitrosylation and oxidation of proteins during cell death, will provide more depth into a
comprehension of cell death in plants.

Author Contributions: Conceptualization, S.M. (Sheikh Mansoorand), O.A.W. and P.A. (Pravej Ala-
mand), writing—original draft preparation S.M. (Sheikh Mansoorand), O.A.W. and P.A. (Pravej Alamand),
J.K.L., S.M. (Sweeta Manhas), N.K., P.A. (Pravej Alam), A.A., writing—review and editing P.A. (Pravej
Alamand), supervision, P.A. (Pravej Alam). All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in review.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
Reactive oxygen species (ROS), Singlet oxygen (1 O2 ), Hydrogen peroxide (H2 O2 ), Superoxide (O2 .− ),
Hydroxyl radical (OH. ), Photosystem (PS), Carbon dioxide (CO2 ), Ribulose-1,5-bisphosphate (RUBP),
Nicotinamide adenine dinucleotide phosphate (NADPH), Catalase (CAT), Mitogen-activated protein
kinase (MAPK), Microbe/pathogen associated molecular patterns (MAMPs/PAMPs), Resistance (R),
Respiratory burst oxidase homolog (RBOH), Polyamine oxidase (PAO), NADPH Oxidases (NOX),
Respiratory burst homolog D (RBOHD), RAC/ROP (Rho-GTPase in plants), Indole 3 butyric acid
(IBR5), Abscisic acid (ABA), Jasmonic acid (JA), Salicylic acid (SA), Ascorbate peroxidases (APXs),
Catalase (CAT), Superoxide dismutase (SOD), Glutathione peroxidases (GPX), Glutathione (GSH),
Ascorbic acid (AA), Phenolic compounds and tocopherols (TOCs), Glutathione reductases (GRs),
Dehydroascorbate reductases (DHARs), Monodehydroascorbate reductases (MDHARs), Ascorbate-
glutathione (AsA-GSH), Gluthation-S-transferase (GSTs), Peroxidases (PER), Respiratory burst oxi-
dase homologs (RBOHs), Gibberellic acid stimulated in Arabidopsis (GASA), Programmed cell death
(PCD), Post-translational modifications (PTMs)

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