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Citation: Timilsina, A.; Dong, W.;
Hasanuzzaman, M.; Liu, B.; Hu, C.
Nitrate–Nitrite–Nitric Oxide
Pathway: A Mechanism of Hypoxia
and Anoxia Tolerance in Plants. Int. J.
Mol. Sci. 2022,23, 11522. https://
doi.org/10.3390/ijms231911522
Academic Editor: Yong-Hwan Moon
Received: 25 August 2022
Accepted: 17 September 2022
Published: 29 September 2022
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International Journal of
Molecular Sciences
Review
Nitrate–Nitrite–Nitric Oxide Pathway: A Mechanism of
Hypoxia and Anoxia Tolerance in Plants
Arbindra Timilsina 1, Wenxu Dong 1, Mirza Hasanuzzaman 2, Binbin Liu 1,3 and Chunsheng Hu 1 ,3 ,*
1Hebei Key Laboratory of Soil Ecology, Center for Agricultural Resources Research, Institute of Genetics and
Developmental Biology, Chinese Academy of Sciences, Shijiazhuang 050021, China
2Department of Agronomy, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Dhaka 1207,
Bangladesh
3
Xiong’an Institute of Innovation, Chinese Academy of Sciences, Xiong’an New Area, Baoding 071700, China
*Correspondence: cshu@sjziam.ac.cn
Abstract:
Oxygen (O
2
) is the most crucial substrate for numerous biochemical processes in plants. Its
deprivation is a critical factor that affects plant growth and may lead to death if it lasts for a long time.
However, various biotic and abiotic factors cause O
2
deprivation, leading to hypoxia and anoxia
in plant tissues. To survive under hypoxia and/or anoxia, plants deploy various mechanisms such
as fermentation paths, reactive oxygen species (ROS), reactive nitrogen species (RNS), antioxidant
enzymes, aerenchyma, and adventitious root formation, while nitrate (NO
3−
), nitrite (NO
2−
), and
nitric oxide (NO) have shown numerous beneficial roles through modulating these mechanisms.
Therefore, in this review, we highlight the role of reductive pathways of NO formation which lessen
the deleterious effects of oxidative damages and increase the adaptation capacity of plants during
hypoxia and anoxia. Meanwhile, the overproduction of NO through reductive pathways during
hypoxia and anoxia leads to cellular dysfunction and cell death. Thus, its scavenging or inhibition is
equally important for plant survival. As plants are also reported to produce a potent greenhouse gas
nitrous oxide (N
2
O) when supplied with NO
3−
and NO
2−
, resembling bacterial denitrification, its
role during hypoxia and anoxia tolerance is discussed here. We point out that NO reduction to N
2
O
along with the phytoglobin-NO cycle could be the most important NO-scavenging mechanism that
would reduce nitro-oxidative stress, thus enhancing plants’ survival during O
2
-limited conditions.
Hence, understanding the molecular mechanisms involved in reducing NO toxicity would not only
provide insight into its role in plant physiology, but also address the uncertainties seen in the global
N2O budget.
Keywords: denitrification; plants; hypoxia and anoxia; nitric oxide signaling; nitric oxide toxicity
1. Introduction
Oxygen (O
2
) deficiency hinders respiration and other biochemical processes essential
for plants’ survival, but extreme events such as heavy precipitation and flooding cause
waterlogging, which directly affects O
2
supply and prevents their growth [
1
]. Moreover,
several other conditions can also lead to hypoxic and anaerobic conditions in well-aerated
tissues of plants. For example, pathogen attacks, tissue exposure to freezing, sulfur dioxide
(SO
2
), ozone, and water deficiencies can cause anaerobic conditions, leading to anaerobic
metabolisms in plant tissues [
2
,
3
]. Abiotic stress such as salt stress can disrupt the symplas-
tic connections between cells, which decreases the permeability of cells to O
2
, resulting in
hypoxia and anoxia [
4
,
5
]. Moreover, under normal conditions, an endogenously generated
O
2
gradient also exists, such that the O
2
concentration may fall below 5% in plant tissues,
such as in seeds, bulk tissues, shoot apical meristems, and roots [6].
Hypoxia and anoxia result in the modification of various normal metabolic paths [
7
].
Thus, they usually inhibit respiration, photosynthesis, nitrogen assimilation, biological
nitrogen fixation, water and nutrient uptake, and stomata closure in plants [
5
,
7
–
11
] through
Int. J. Mol. Sci. 2022,23, 11522. https://doi.org/10.3390/ijms231911522 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2022,23, 11522 2 of 24
a reduced adenosine triphosphate (ATP) concentration, nicotinamide adenine dinucleotide
(NAD
+
) and nicotinamide adenine dinucleotide hydrogen (NADH) ratio (NAD
+
/NADH),
and cell viability [
12
]. Meanwhile, the accumulation of reactive oxygen species (ROS)
and reactive nitrogen species (RNS) is triggered, which severely damages the cell compo-
nents [
13
]. Moreover, during hypoxia and anoxia, a drop in pH causes cytoplasmic acidosis
which affects numerous metabolic activities that may even contribute to plant death [
14
].
Overall, hypoxia and anoxia have numerous deleterious effects on plant metabolism (Fig-
ure 1). The effects of O
2
deficiency could be more severe to hypoxia- and anoxia-intolerant
plants as compared to tolerant plants. For example, O
2
stress directly reduces germination
rate and coleoptile growth in barley, oat, and rice, while growth is more pronounced in
anoxia-tolerant rice than in barley and oat [1,15].
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 2 of 24
Hypoxia and anoxia result in the modification of various normal metabolic paths [7].
Thus, they usually inhibit respiration, photosynthesis, nitrogen assimilation, biological
nitrogen fixation, water and nutrient uptake, and stomata closure in plants [5,7–11]
through a reduced adenosine triphosphate (ATP) concentration, nicotinamide adenine di-
nucleotide (NAD+) and nicotinamide adenine dinucleotide hydrogen (NADH) ratio
(NAD+/NADH), and cell viability [12]. Meanwhile, the accumulation of reactive oxygen
species (ROS) and reactive nitrogen species (RNS) is triggered, which severely damages
the cell components [13]. Moreover, during hypoxia and anoxia, a drop in pH causes cy-
toplasmic acidosis which affects numerous metabolic activities that may even contribute
to plant death [14]. Overall, hypoxia and anoxia have numerous deleterious effects on
plant metabolism (Figure 1). The effects of O2 deficiency could be more severe to hypoxia-
and anoxia-intolerant plants as compared to tolerant plants. For example, O2 stress di-
rectly reduces germination rate and coleoptile growth in barley, oat, and rice, while
growth is more pronounced in anoxia-tolerant rice than in barley and oat [1,15].
Figure 1. Possible causes of hypoxia and anoxia, their consequences, and defense mechanisms in
response to O2 deficiency. Red arrows represent negative effects to plants, while green ones repre-
sent positive effects.
To survive O2 deficiency, plants use numerous strategies through biochemical, ana-
tomical, and morphological changes (Figure 1). However, the accumulation of ethanol and
lactic acid (major products of the fermentation process) are toxic [16]. Moreover, antioxi-
dant defense systems could also be a limiting factor if the stress is present for a longer
time or beyond the tolerance capacity. This suggests that if anaerobic processes proceed
for a longer time, the ultimate fate of plants is death. Along with metabolic changes, plant
adaptation mechanisms can improve tissue O2 status. A number of mechanisms have been
reported to help plants to improve O2 status during soil waterlogging conditions. For ex-
ample, O2 distribution from aerial parts to roots is facilitated by the formation of
aerenchyma [17,18], and its formation is faster in flood-tolerant than intolerant plants
[18,19]. Similarly, adventitious root formation can also improve the O2 status of plants
during waterlogging conditions [20]. Meanwhile, the balanced production of ROS and
Figure 1.
Possible causes of hypoxia and anoxia, their consequences, and defense mechanisms in
response to O
2
deficiency. Red arrows represent negative effects to plants, while green ones represent
positive effects.
To survive O
2
deficiency, plants use numerous strategies through biochemical, anatom-
ical, and morphological changes (Figure 1). However, the accumulation of ethanol and
lactic acid (major products of the fermentation process) are toxic [
16
]. Moreover, antioxidant
defense systems could also be a limiting factor if the stress is present for a longer time or
beyond the tolerance capacity. This suggests that if anaerobic processes proceed for a longer
time, the ultimate fate of plants is death. Along with metabolic changes, plant adaptation
mechanisms can improve tissue O
2
status. A number of mechanisms have been reported
to help plants to improve O
2
status during soil waterlogging conditions. For example, O
2
distribution from aerial parts to roots is facilitated by the formation of aerenchyma [
17
,
18
],
and its formation is faster in flood-tolerant than intolerant plants [
18
,
19
]. Similarly, ad-
ventitious root formation can also improve the O
2
status of plants during waterlogging
conditions [
20
]. Meanwhile, the balanced production of ROS and RNS and an increase in
antioxidant activities can enhance tolerance to hypoxia and anoxia in plants [21].
Nitric oxide (NO), a widely recognized signaling molecule, plays an important role
in hypoxia and anoxia tolerance in plants [
22
,
23
]. Not only NO but also nitrate (NO
3−
),
Int. J. Mol. Sci. 2022,23, 11522 3 of 24
nitrite (NO
2−
), and nitrate reductase (NR, EC 1.6.6.1) play a similar role in plants during
O
2
deficiency [
24
]. This suggests that tolerance to O
2
deficiency is due to the reductive
pathways of NO formation. However, numerous studies indicate that O
2
deficiency, as well
as other stresses, can trigger NO formation [
23
–
25
]. Meanwhile, a higher concentration of
NO could be cytotoxic, leading to the accumulation of ROS and other RNS that would lead
to nitro-oxidative stress [
25
,
26
]. Nitric oxide could promote [
27
] or inhibit [
28
] ethylene
biosynthesis, a key phytohormone for plants’ survival during O
2
limitation, while the
latter case is mediated through the S-nitrosylation of methionine adenosyltransferase
(MAT1) [
28
]. Phytohormones such as salicylic acid (SA), jasmonic acid, and abscisic acid
(ABA) reduce oxidative stresses and enhance the activities of antioxidants during stress
conditions [
29
,
30
]. However, NO is reported to inhibit the activities of antioxidants, as well
as proteins involved in regulating phytohormones through S- nitrosylation [
30
–
32
], thus,
again, increasing nitro-oxidative stress in plants.
Thus, there should be a fine regulation of these signaling molecules (NO, ROS, and
other RNS) for beneficial roles. The key to surviving during hypoxia and anoxia depends
upon mechanisms that could lessen the harmful effects of nitro-oxidative damages by
increasing the activities of adaptation mechanisms. Therefore, understanding the reductive
pathways of NO formation along with NO scavenging mechanisms would provide insight
into the mechanisms involved in lessening the effects of O
2
deprivation. Hence, the main
aim of this review is to highlight the role of reductive pathways of NO formation, while
emphasizing the NO scavenging mechanisms that could reduce the nitro-oxidative stress
and increase the hypoxia and anoxia tolerance in plants.
2. Pathways of NO Formation during Hypoxia and Anoxia
Various pathways of NO formation in plant cells have been documented, and they
have been categorized into oxidative and reductive pathways. Oxidative pathways are
oxygen-dependent, involving L-arginine, polyamine, and hydroxylamine [
33
]. The reduc-
tive pathways of NO formation occur during low O
2
and are dependent on NO
3−
, NR,
NO
2−
, plasma membrane NR, plasma membrane-bound nitrite reductase (PM NiNOR),
xanthine oxidoreductase in plant peroxisomes, photosynthetic-electron-transport-chain-
dependent NO
2−
reduction in chloroplasts, and mitochondrial electron transport chains
(ETCs) such as cytochrome bc
1
complex (complex III, EC 1.10.2.2), cytochrome c oxidase
(CcO, EC 1.9.3.1), and alternative oxidase (AOX, EC 1.10.3.11) in mitochondria [
33
,
34
].
In Chlamydomonas reinhardtii, NR, together with nitric-oxide-forming nitrate reductase
(NOFNiR), reduces NO
2−
to NO [
35
]. Moreover, NO
2−
can be reduced to NO in acidic
pH without the involvement of any enzyme. NO production pathways during O
2
defi-
ciency and other stresses would be different. For example, salt stress can increase both
oxidative pathways (l-arginine-dependent) [
36
] as well as the reductive pathways of NO
production [
37
]. However, during O
2
deficiency, NO is produced through the reductive
pathways [
33
]. Interestingly, this occurs not only during O
2
deficiency but many other
biotic- and abiotic-stress-induced reductive pathways of NO formation. For example, salin-
ity stress, water deficiency, UV radiation, freezing, pathogen attacks, and wounding can
trigger NO production in plants [
38
–
43
], which could be due to the fact that these stresses
could lead to hypoxia and anoxia in plant tissues, while its formation could be a defense
strategy to survive harsh conditions.
Nitric oxide is formed in various cell compartments such as the cytosol, apoplasts,
chloroplasts, peroxisomes, and mitochondria of plants through enzymatic or non-enzymatic
pathways [
33
]. Nitric oxide production in various compartments of plant cells has nu-
merous functions. For example, NO formed in chloroplasts can prevent the oxidation of
chloroplastic lipids and proteins, while NO-mediated peroxynitrite (ONOO
−
) production
may result in its damage [
44
]. Similarly, NO formed in mitochondria can protect its compo-
nents, while NO-mediated ONOO
−
production causes mitochondrial dysfunction [
45
]. As
O
2
limitation, as well as other stresses, leads to a higher level of NO formation through the
Int. J. Mol. Sci. 2022,23, 11522 4 of 24
reductive pathways, understanding the possible mechanisms for maintaining the optimum
level of NO is also essential and is discussed in later sections.
3. Role of Nitrate and Nitrate Reductase (NR) during Hypoxia and Anoxia Tolerance
Nitrate is not only an important form of nitrogen (N) source to plants but also a
signaling molecule [
46
]. It is usually a major form of N in aerobic soil, and its uptake by
plant roots is achieved through NO
3−
transporters [
47
]. After being uptaken by roots, NO
3−
is reduced to NO
2−
by an enzyme called NR in the cytosol or plasma membrane or stored
in the vacuole or transported to shoots and leaves for subsequent reduction [
16
]. Under
normoxia, NO
2−
is transported to plastids/chloroplasts and is reduced to ammonium
(NH
4+
) by nitrite reductase (NiR, EC 1.7.7.1). Then, glutamine synthetase/glutamate-
oxoglutarate aminotransferase (GS, EC 6.3.1.2)/GOGAT, EC 1.4.1.13) assimilates NH
4+
into amino acids. However, during hypoxia and anoxia, the NO
3−
or NH
4+
assimilation
path to amino acid as well as NO
3−
transport to the aerial parts is greatly reduced [
48
].
For example, O
2
deficiency decreases NO
3−
and NH
4+
assimilation and N incorporation
into amino acids in various plant species as compared to normoxia [
49
–
51
]. Although N
incorporation into amino acids is inhibited during O
2
deficiency, several pieces of research
have shown that NR is highly activated and NO
3−
is reduced to NO
2−
[
52
]. Interestingly,
NO
3−
consumption by soybean plants in hydroponics systems was higher during hypoxia
than normoxia [
48
], which suggests that more NO
3−
is metabolized during O
2
-limited
conditions in plant cells. Therefore, most of the derivatives of NO
3−
might be lost to the
environment in the form of gases from plants during O2limitation.
Several previous studies have shown that NO
3−
and NR are beneficial for hypoxia
and anoxia tolerance. Germinating seeds generally experience hypoxic and anoxic condi-
tions [
53
–
55
] due to the compaction and hindrance of O
2
diffusion by the outermost layers
of seeds [
56
]. Studies have reported that NO
3−
is beneficial during seed germination. For
example, supplementation or priming with NO
3−
increases the viability of germination
in seeds of various plants [
57
–
60
]. Light and temperature influence seed germination,
while NO
3−
can reduce the dependency on environmental factors such as light [
58
,
61
]
and temperature [
62
] during germination. Moreover, NO
3−
can promote germination in
seeds during salt, metal, and heat stresses [
63
–
65
]. The mechanisms of seed germination
by NO
3−
might be due to NO production in cytosol and mitochondria through the reduc-
tive pathways [
55
]. Similarly, NO
3−
has been shown to increase activities of antioxidant
enzymes such as catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1)
during the germination process [
59
], which could scavenge ROS, thus preventing oxidative
damage and promoting germination. Seed germination is promoted during conditions
with a lower level of ABA [
66
] and a higher level of gibberellic acid (GA) [
67
], while NO
3−
supplementation leads to the upregulation of the ABA catabolic gene CYP707A2 and GA
biosynthesis gene GA20ox1 [
64
], thus promoting seed germination by decreasing ABA and
increasing GA levels. Although ethylene is widely reported to promote seed germination,
the role of NO
3−
on its biosynthesis is unclear. Endogenous NO
3−
levels in germinating
seeds drop significantly during the first 24 h post-imbibition, and the role of NO
3−
and
NR activity in anaerobic seed germination depends on NADH and NADPH [
68
], which
could be due to the fact that NO
3−
serves as an alternate electron acceptor [
69
], similar to
bacterial denitrification [
70
]. This is supported by the fact that NO
3−
and O
2
limitation
induces high levels of both NO and N2O emissions from plants [24].
Waterlogging reduces several nutrients in plants, affecting plant metabolism [
71
], while
the supplementation of NO
3−
increases the uptake of nutrients such as N, P, Fe, and Mn [
72
].
Nitrate can improve cytoplasmic acidification caused by anoxia in plants
[73,74]
while de-
creasing fermentative enzymes such as lactate dehydrogenase (LDH, EC 1.1.1.27), pyruvate
decarboxylase (PDC, EC 4.1.1.1), and alcohol dehydrogenase (ADH, EC 1.1.1.1) [
75
]. Lower
levels of lactate and ethanol in plant roots [
10
,
75
] and an increase in the ATP level were
observed in NO
3−
-treated plants during waterlogging [
75
], which suggest that NO
3−
is
highly beneficial to reducing toxic metabolites while increasing the energy status of water-
Int. J. Mol. Sci. 2022,23, 11522 5 of 24
logged plants. Antioxidants such as SOD, CAT, ascorbate peroxidase (APX, EC 1.11.1.11),
and guaiacol peroxidase (POD, EC 1.11.1.7) remove O
2−
and H
2
O
2
[
76
,
77
]. Nitrate-fed
plants show increased activities of antioxidants such as SOD and CAT, APX, and POD,
thereby decreasing the level of H
2
O
2
and O
2−
[
21
,
78
], thus increasing tolerance to hypoxia
and anoxia during waterlogging. Speedy recovery following re-oxygenation is equally
important for plant growth, while NO
3−
has been shown to be beneficial during hypoxia
and subsequent re-oxygenation by inducing antioxidant systems in plants [79].
Hypoxia and anoxia in roots caused by flooding decrease chlorophyll content in the
leaves of plants, thus decreasing the plant biomass and photosynthesis rate [
11
]. Nitrate
is more beneficial in terms of biomass, net photosynthesis rate, chlorophyll, and protein
content as compared to NH
4+
and glycine [
21
,
80
]. Moreover, the concentration of metabo-
lites such as sucrose,
γ
-aminobutyrate, succinate, and nucleoside triphosphate are reduced
significantly in the absence of NO
3−
during hypoxia in maize root [
73
]. Alanine amino-
transferase (AlaAT, EC 2.6.1.2), via the reversible conversion of pyruvate and glutamate to
alanine and 2-oxoglutarate, is involved in carbon and nitrogen metabolism [
81
]. The foliar
spraying of NO
3−
during waterlogging increases AlaAT and GOGAT activities along with
an increase in amino acid in plants [
82
], suggesting that NO
3−
is involved in regulating
both glycolysis and the TCA cycle during O2deficiency.
The nodulation of soybean plants with symbiotic bacteria is beneficial for plant growth.
However, during hypoxia, non-nodulated soybean plants supplied with NO
3−
have shown
many beneficial effects such as more antioxidant and less oxidative damage through
reduced ROS and H
2
O
2
production as compared to nodulated soybean plants without
NO
3−
[
79
]. Similarly, nodulated soybean plants exposed to hypoxia decreased in biomass
by 34%, while non-nodulated plants supplemented with NO
3−
only decreased in biomass
by 12% [
83
]. Moreover, plant NR is involved in nitrogen fixation, energy generation,
maintaining cytosolic pH, and the metabolism of carbon and nitrogen in nodules during
hypoxia in plant–microbe symbiosis [
84
–
86
]. This suggests that during hypoxia, NO
3−
and
NR are more beneficial than the symbiotic relationship alone.
Phytoglobins (Pgbs) play an important role during hypoxia and anoxia tolerance in
plants. Numerous studies have reported that the increase in Pgbs during hypoxia and
anoxia [
87
,
88
] and its expression are related to survival during O
2
-limited conditions in
plants [
87
]. Nitrate nutrition during hypoxia has been beneficial in the overexpression
of Pgbs [
88
]. During hypoxia and anoxia, NO
3−
and NR are involved in ATP produc-
tion through reductive pathways and the phytoglobin-NO respiration cycle [
86
,
89
]. This
phytoglobin-NO respiration cycle helps in maintaining cellular bioenergetics by preventing
the over-reduction of NAD and NADH [
90
]. Ethylene is responsible for the stability of the
group VII ethylene response factor which could lead to the induction of several hypoxic
genes [
91
], while NO
3−
nutrition during hypoxia has led to the activation of the 4.5-fold
induction of the ETR1 gene (At1g66340.1) responsible for ethylene production in plants [
88
].
The role of ethylene during hypoxia and anoxia tolerance could be due to its role in NO
scavenging by inducing Phytoglobin 1 (Pgb1) mRNA levels, as ethylene does not increase
NR activities during O
2
-limited conditions [
92
]. Interestingly, NO
3−
is the substrate re-
sponsible for NO production, and it also plays a role in NO scavenging through inducing
Pgb1 as well as ethylene biosynthesis, suggesting a more beneficial role of NO3−.
Redox imbalance during hypoxia and anoxia directly affects cellular metabolisms [
93
].
Various studies have reported that NO
3−
supplementation to hypoxic and anoxic plant
tissues can improve the redox state [
16
,
94
,
95
]. For example, NO
3−
and NR maintain redox
balance during hypoxia in cucumber (Cucumis sativus L.) [
12
]. Mitochondria are the most
important organelles for survival, and their functionality is more crucial under hypoxia
and anoxia [
96
]. In the absence of O
2
, NO
3−
could act as a terminal electron acceptor in
plants [
69
], while it also plays an important role in maintaining mitochondrial ultrastructure
during anoxia. For example, in the absence of NO
3−
, cristae disappear, the matrix loses its
electron density, and after a few hours, mitochondria completely degrade [
69
], while its
presence protects the ultrastructure of mitochondria during hypoxia and anoxia [
97
]. In
Int. J. Mol. Sci. 2022,23, 11522 6 of 24
humans, NO
3−
can protect against ischemia/reperfusion injury, reduce blood pressure, and
improve oxidative phosphorylation efficiency (P/O ratio), indicating a decrease in proton
leakage and membrane potential is distributed towards ATP synthesis in mitochondria [
98
].
Nitrate reduction via NR can delay cell death during hypoxia and delay the anoxic
symptoms in plants [
99
], while its inhibition can significantly disturb the growth [
95
].
Tobacco (Nicotiana tabacum) mutant plants lacking NR reductase are more sensitive to
O
2
deprivation as compared to wild types by showing symptoms of rapid wilting, more
ethanol and lactate production, and less ATP generation [
94
], suggesting the role of NO
3−
is due to its reduction to NO
2−
. NR plays a role in the maintenance of energy status for
nitrogen fixation under O
2
-limited conditions in Medicago truncatula nodules [
100
]. The
use of NR inhibitors in the root system of nodulated alfalfa (Medicago sativa L.) results
in a significant decrease in the ATP/ADP ratio under flooding and salinity stresses [
5
].
Waterlogging significantly degrades membrane lipids [
101
], while NO
3−
and NR activity
can delay the anoxia-induced degradation of membrane lipids in plant cells [
102
]. Higher
expression of NR in cucumber (Cucumis sativus) than tomato (Lycopersicon esculentum) was
associated with a high tolerance of hypoxia in the roots [
103
]. During hypoxia and anoxia,
NR plays an important role in plant biology by regulating NO production by supplying
electrons to NOFNiR and truncated hemoglobin [
104
]. The regulation of NO is critical,
as it is a signaling and also toxic molecule if it is accumulated in a higher amount in
a cell [
105
]. Overall, both NO
3−
and NR are involved in hypoxia and anoxia tolerance
with numerous benefits, which suggests that NO
2−
is also involved in the mechanisms.
However, long-term O
2
limitation would affect the NR acclivity, thus, again, questioning
plants’ survival during O
2
-limitation conditions. For example, the NR level increases
during O
2
limitation conditions, while NR-mRNA remains constant during the early hours
of O
2
limitation and decreases after 48 h [
99
], suggesting long-term O
2
limitation affects
its activity. Moreover, NO, which is produced by NR itself, also decreases the level of
NR protein through posttranslational modifications and ubiquitylation by affecting amino
acids involved in binding the essential flavin adenine dinucleotide (FAD) and molybdenum
cofactors [
35
,
106
]. Therefore, O
2
limitation and a higher level of NO formation would affect
NR activity after long-term hypoxia and anoxia, thus, again, affecting plants’ survival.
Moreover, a higher concentration of NO
3−
is reported to affect plant growth through the
increased production of NO, thus increasing lipid peroxidation and the H
2
O
2
level [
107
].
This dose-dependent effect of NO
3−
might be due to the fact that beyond a certain level of
its concentration, there would be more NO production through the reductive pathways,
which could not be scavenged effectively, thus promoting ONOO
−
formation, causing
harmful effects.
4. Role of Nitrite during Hypoxia and Anoxia Tolerance
A well-known pathway of NO
2−
metabolism in plants is its assimilation to amino
acids through reduction to NH
4+
. However, during O
2
deprivation, the assimilatory
pathway is inhibited, and NO
2−
is either accumulated in the cytoplasm [
49
] or reduced to
NO by the NR in the cytoplasm or transported to mitochondria for reduction [
108
]. This
is further supported by the fact that NiR is inhibited during O
2
-limited conditions [
49
].
Although NO
2−
assimilation to amino acids is significantly reduced during hypoxia and
anoxia, NR is activated, and the NO
2−
level increases [
16
,
52
]. Studies suggest that NO
2−
reduction to NO through reductive pathways is beneficial during hypoxia and anoxia [
108
].
Similar to NO
3−
, NO
2−
can promote seed germination in plants [
55
,
57
,
109
]. Moreover,
thermo-dependency during seed germination was lowered in the presence of NO
2−
[
61
].
During low O
2
levels in mitochondria, NO
2−
can regulate the surrounding O
2
concentra-
tion through the production of NO [
53
]. Exogenous NO
2−
can also reduce both ethanol
and lactate production [
110
] and can minimize the acidification of cytoplasm in plants
during hypoxia and anoxia [
74
]. Similarly, the role of NO
2−
in the protection of mito-
chondrial structures and functions has been well documented. NO
2−
supplementation
during O
2
-limited conditions to the mitochondria isolated from roots of pea (Pisum sativum)
Int. J. Mol. Sci. 2022,23, 11522 7 of 24
shows better mitochondrial integrity, the energization of the inner mitochondrial mem-
brane, increased ATP synthesis, and decreased production of ROS and also decreased
lipid peroxidation [
111
]. Hypoxia and anoxia can degrade the activities of complex I [
112
],
while NO
2−
supplementation can result in its higher levels and activities [
111
]. The role of
NO
2−
in hypoxia tolerance in humans and animals has been well documented [
113
,
114
].
It could be through its reduction to NO, as hypoxia and anoxia trigger NO
2−
reduction
to NO. However, a higher concentration of NO
2−
can lead to membrane damage, lipid
peroxidation, protein oxidation, mutation, DNA damage, and cell death [
115
], which could
be through a higher level of NO production. So, for its beneficial role, its concentration
should be regulated.
5. Role of Nitric Oxide during Hypoxia and Anoxia Tolerance
The role of NO in plant physiology has been described by numerous researchers.
The reductive pathway of NO formation in plants is reported to be beneficial in plants
as it promotes seed germination, increases biomass and root formation, increases energy
status during O
2
limitation, promotes tolerance to various biotic and abiotic stresses, and
promotes the induction of different defense-related genes, and many others, as tabulated in
Table 1.
Table 1.
Role of NO
3
-NO
2
-NO pathway during oxygen-limited conditions and other stresses on
plant defense mechanisms.
Activities/Defense Mechanisms Conditions References
NO3−Maintains photosynthesis and transpiration Waterlogging [21]
NO3−Protection of mitochondrial ultrastructure for a
longer time Anoxia [97]
NO3−Maintains membrane stability Hypoxia [102]
NO3−
Higher activities of antioxidant enzymes such as
SOD, APX, CAT, glutathione reductase (GR, EC
1.8.1.7), and guaiacol peroxidase (GPOD, EC 1.11.1.7)
Hypoxia [79]
NO3−Increases the various nutrient contents Waterlogging [72]
NO3−Increases seed germination rate by regulating the
ABA level Normoxia [66]
NR inhibition Growth is disturbed Waterlogging [94,95]
NO3−Increases ATP synthesis while decreasing
fermentation Waterlogging [75]
NO3−
Maintains the level of metabolites such as sucrose,
alanine,
γ
-aminobutyrate, lactate, and succinate and
decreases fermentation
Waterlogging [73]
NO3−
Increases in CO
2
assimilation, stomatal conductance,
transpiration rate, and shoot biomass Waterlogging [78]
NO3−
UV-radiation tolerance by reducing H2O2and
malondialdehyde (MDA) and increasing plants’
height and biomass
UV stress [116]
NO3−and NR Delay wilting and anoxia symptoms Anoxia [99]
NR-deficient mutant plant
Produces less NO that is more susceptible to
bacterial and fungal attack through decreasing
hypersensitive response
Pathogen attack [117]
NO2−Decreases fermentation that helps to reduce the
toxicity of fermentative metabolites Hypoxia [10]
NO3−and NO2−Improves cytoplasmic acidification Hypoxia and Anoxia [73,74]
NO2−ATP synthesis through mitochondria ETCs Anoxia [118]
Int. J. Mol. Sci. 2022,23, 11522 8 of 24
Table 1. Cont.
Activities/Defense Mechanisms Conditions References
NO2−Protects mitochondrial structure and functions Hypoxia [111]
NR-dependent NO
production
Defense against pathogen through rapid
development of hypersensitive response and
lessening the effects of clorotic lesions and bacterial
infection
Pathogen attack [40,119]
NR-dependent NO
production
Involved in cold acclimation and freezing tolerance
through reductions in electrolyte leakage Cold stress [39]
NO
Decreases the mitochondrial oxidative damages
through decreased ROS content and maintained the
structure and function of mitochondria through
increasing mitochondrial antioxidants enzymes,
improving mitochondrial Ca2+ homeostasis,
promoting genes related to C-repeat binding factors
(CBFs), while reducing the peroxidation of
mitochondrial fatty acids
Cold stress [120,121]
NO
Maintains quality, delays ripening, and enhances
resistance to pathogens through increasing the
activities of antioxidants, gene regulation, and
suppressing ethylene production
Postharvest storage [122]
NR-dependent NO
production
Aluminum-induced ROS and lipid peroxidation are
reduced, while it improves root growth during the
stress through the regulation of
ascorbate–glutathione cycle
Metal stress [123,124]
NR-dependent NO
production
Copper tolerance through enhanced antioxidant
activities Metal stress [125]
NO
Improved seed germination through upregulation of
α
-amylase, protease, enzymes of N assimilation, and
antioxidants
Metal stress [126]
NR-dependent NO
production
Salt tolerance by balancing redox and ions, reducing
ROS, and increasing antioxidants Salt stress [127]
NO Increases activities of antioxidants and proline
content Salt stress [128]
NR-dependent NO
production
The rapid accumulation of UV-absorbing substances
such as flavonoids UV stress [41,129]
NR-dependent NO
production
Higher photosynthetic rates and stomatal
conductance, and less ROS accumulation due to
higher activities of various antioxidants
Drought stress [43]
NO Improved photosynthesis activities and promotes
growth Drought stress [130]
NR-dependent NO formation
and induction of
non-symbiotic hemoglobin
Root elongation through the activities of actin
cytoskeleton and hormonal signaling Normoxia [131]
NR-dependent NO
production
Releases tuber dormancy and sprouting via the
expression of genes involved in ABA catabolism Normoxia [132]
NO3−dependent NO
production
Regulation of lateral root and seminal root growth
by regulating auxin transport, while lateral root
formation increases N uptake capacity during
partial N availability
Normoxia [133,134]
NO2−dependent NO
production Regulates O2concentration and postpone anoxia Hypoxia [53]
Int. J. Mol. Sci. 2022,23, 11522 9 of 24
Table 1. Cont.
Activities/Defense Mechanisms Conditions References
NO2−and NO
Accelerates germination through decreasing lipid
peroxidation and DNA fragmentation in
germinating seeds
Physiological hypoxia [55]
NO
Decreases cell membrane injuries and increases
stomatal conductance and transpiration rate as
compared to the control
Waterlogging [135]
NO3−, NO2−and NO Breaks dormancy in seeds through NO signaling Normoxia [109]
NO
Increases biomass and lint yield of cotton plants
through reduced lipid peroxidation, the expression
of waterlogging tolerance-related genes, and
increasing photosynthesis process
Waterlogging [136]
NO Enhances adventitious root formation Waterlogging [20]
NO
Regulates genes belonging to phytohormones,
Cytochrome P450 encoding genes (CYP72A14 and
CYP707A3) that regulate ROS and genes related to
cell wall synthesis, modification, and degradation
Hypoxia [137]
Similar to NO
3−
and NO
2−
, NO also stimulates germination in various plants species
in a dose-dependent manner, i.e., low to medium NO has a positive effect, while a higher
concentration inhibits germination [
138
–
141
]. The mechanism involved in seed germination
by NO could be due to its capacity to reduce respiration rates and ROS levels while
increasing carbohydrate metabolism and the level of amino acids and organic acids in
germinating seeds [
55
]. The
α
-amylase (EC 3.2.1.1) activities of rice seed germination in
the flooded condition are directly linked to seedling survival [
142
], while NO and GA can
induce the activity of
α
-amylase [
143
]. However, the increase in activities of
α
-amylase by
NO is time-dependent, such that at an early hour, it increases the activities, while prolonged
NO exposure strongly reduces the activities [
55
]. This time-dependent role of NO could
be due to the fact that prolonged exposure to NO could accumulate RNS which inhibit its
activity. NO is involved in controlling seed dormancy through inducing the degradation of
the ABI5 protein, thus enhancing ABA catabolism [
144
] while also increasing antioxidant
enzymes [
132
,
141
]. However, a high level of NO can be toxic to cells, as it can inhibit
mitochondrial respiration irreversibly [
105
] as well as inhibit antioxidants enzymes [
145
],
which could explain the mechanisms of inhibiting germination by a higher level of NO.
NO production in plant cells during hypoxia enhances the survival rate [
146
]. During
waterlogging conditions, the application of NO donor increases leaf area, plant biomass,
harvest index, lint yield, and boll number in the cotton plant [
136
]. Similarly, the net photo-
synthetic rate and chlorophyll content increase, and MDA, H
2
O
2
, ADH, and PDC content
decrease [
136
]. The role of NO in increasing the net photosynthetic rate and chlorophyll
content could be due to its role in inhibiting the transcriptional activation of chlorophyll
breakdown pathway genes such as SRG, NYC1, PPH, and PAO [
147
]. Similarly, during
waterlogging conditions, NO influences both the morphological and physiological char-
acteristics of maize seedlings such that it increases height, dry weight, and antioxidant
activities while decreasing MDA content and the ion leakage ratio [
148
]. The reductive
pathway of NO production is involved in maintaining leaf shape and size in plants by in-
creasing the cell size, chlorophyll a/b contents, antioxidant enzymatic activity, homeostasis
of ROS [149], and root elongation [131].
The mechanisms of hypoxia tolerance by NO are several. For example, NO improves
H
2
S accumulation in maize seedling roots, which increases antioxidant defense, leading to
the removal of excess ROS [
146
]. Moreover, during hypoxia and anoxia, NO is involved
in ATP production through mitochondrial ETCs and the phytoglobin-NO cycle [
16
], thus
increasing energy status. During hypoxia, NO production and the fine regulation of ROS
Int. J. Mol. Sci. 2022,23, 11522 10 of 24
and NO can slow down the respiration rate while preventing tissues from anoxia [
53
].
Nitric oxide could induce the expression of alternative oxidase (AOX) during various stress
conditions [
150
], while its expression is associated with less superoxide generation and lipid
peroxidation during O
2
limitation conditions, while AOX also prevents nitro-oxidative
stress during reoxygenation [151].
During plant–microbial symbiosis, the enzyme of nitrogen fixation, i.e., nitrogenase,
is only stable and functional in O
2-
limited conditions [
152
]. In such symbiotic interaction,
plant NR and mitochondrial ETCs are involved in NO production, while excess and low
NO inhibit the nodule establishment [
85
], suggesting that NO should be regulated in the
symbiotic relationship between plants and microbes. The nitrate-NO respiration process
in root nodules of Medicago truncatula plays a role in the maintenance of the energy status
required for nitrogen fixation [100].
Calcium ion reduces the level of ROS and increases the antioxidant enzymes in mito-
chondria during hypoxia by improving metabolism and ion transport in plants, thereby
increasing hypoxia tolerance [
153
]. Similarly, exogenous calcium application can increase
the biomass, net photosynthesis, stomatal conductance, and efficiency of photosystem
II during hypoxia stress in plants [
154
]. NO can regulate Ca
2+
in plant cells [
155
]. For
example, plant cells treated with NO donors are reported to have a fast increase in cytosolic
Ca
2+
concentration, which was strongly reduced when treated with NO scavengers [
156
].
The mechanism involved in this regulation of Ca2+ could be that NO can increase the free
cytosolic Ca
2+
concentration by activating plasma membrane Ca
2+
channels and induc-
ing plasma membrane depolarization [
156
]. However, a higher concentration of NO is
also reported to inhibit the cytosolic Ca
2+
in human cells [
157
], suggesting NO should be
regulated for its beneficial role.
If a plant is exposed to O
2
deficiency for a prolonged period, the ultimate fate of the
plant will be death. So, the mechanisms that could improve the O
2
status of waterlogged
plants would only benefit the plant to survive, while NO formation is also highly beneficial
for improving O
2
status through various mechanisms. For example, adventitious root
formation increases plant resistance to waterlogging by increasing the inward diffusion of
O
2
[
20
] or even participating in photosynthesis, thus improving O
2
status [
158
], while NO
is reported to play a role on its formation during waterlogging [
20
]. Similarly, aerotropic
roots can be originated in lateral roots that emerge above the water surface if waterlogging
lasts for a prolonged period [
159
], while NO generation through reductive pathways is
involved in lateral root and seminal root elongation in plants [
133
] that could facilitate
the O
2
supply together with aerotropic roots. Aerenchyma formation allows O
2
diffusion
from aerated to waterlogged parts of plants, while NO also plays a role in aerenchyma
formation in plants [
27
], thus improving the O
2
status. Nitric oxide as well as ethylene
are involved in programmed cell death and aerenchyma formation during O
2
-limitation
conditions [
27
,
160
]. Nitric oxide formed through reductive pathways induces the expres-
sion of aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase
(ACO) genes responsible for ethylene synthesis [
27
]. Recently, it has been reported that
auxin is involved in ethylene-dependent aerenchyma formation such that the use of an
auxin transport inhibitor abolished the arenchyma formation [
160
]. Meanwhile, during O
2
limitation conditions, NO donors could induce the upregulation of the auxin transporter
PIN2 gene [
137
], suggesting the diverse roles of NO in regulating aerenchyma formation in
plants.
6. Adverse Effects of Nitric Oxide and Role of Nitric Oxide Scavenging on Hypoxia
and Anoxia Tolerance
It is clear that NO, as the end product of the NO
3
-NO
2
-NO pathway, plays numerous
beneficial roles during hypoxia and anoxia tolerance in plants. However, to be beneficial, the
concentration of NO plays a critical role, while hypoxia and anoxia trigger NO production,
which can be lethal to cells [
105
]. Some of the adverse effects of NO are summarized in
Table 2. Moreover, oxidative stress caused by O
2
limitation and the overproduction of NO
Int. J. Mol. Sci. 2022,23, 11522 11 of 24
during various stresses could damage major components of mitochondria [
112
,
161
] and
inhibit antioxidants systems, thus accumulating ROS and RNS [
22
]. RNS, if accumulated
more, could exacerbate more damage than ROS by triggering free radical peroxidation [
162
].
Increased RNS and ROS production could lead to retrograde signaling to the nucleus to
regulate gene expressions [
163
]. Nitric oxide, through the formation of RNS, could lead
to mutation, DNA damage, and cell death [
161
,
164
]. So, for the longer survival of a cell
during hypoxia and anoxia, the NO produced RNS should be scavenged efficiently.
Table 2.
Adverse effects of a higher level of NO in plants. The high level of NO was achieved through
a higher dose of NO donor or using NO-overproducing mutants or hypoxia plus NO donors.
Effects of Higher Level of NO References
Decreases the root growth through DNA damage, induces cell cycle arrest and inhibits primary root
growth by affecting root apical meristem activity and cell elongation. [165,166]
Delayed flowering, retarded root development, and reduced starch granule formation through
S-nitrosylation modification. [167]
Cell death through increased electrolyte leakage, cell wall degradation, cytoplasmic streaming, and
DNA fragmentation. [27]
Decreases the expression of cyclins (CYC) and Cyclin-Dependent Kinases (CDKs), resulting in the
downregulation of cell cycle progression. [168]
NO can generate peroxynitrite, which is a mediator of cytochrome c loss, protein oxidation and
nitration, lipid peroxidation, mitochondrial dysfunction, damage DNA, and cell death. [26,169]
NO can inhibit antioxidants such as catalase, glutathione peroxidase (GPX), and ascorbate peroxidase
in a reversible way and peroxynitrite in an irreversible way. [145,170]
NO can change the redox state and promote cell death. [33]
Inhibits lateral and primary root growth through reduced cell division and the expression of the
auxin reporter markers DR5pro:GUS/GFP.[166,171]
Inhibits growth of tobacco plants through peroxynitrite formation and tyrosine nitration. [172]
Inhibits seed germination, while the scavenging of NO alleviates the effect. [139]
Inhibits the shoot growth and decreases the chlorophyll contents of the plants. [173,174]
It is clear that NO scavengers work differently in plants. For example, the use of
NO scavengers during low NO production have negative effects on plant growth [
133
],
while during high NO production, the same NO scavengers have positive effects [
166
].
A similar role of NO has been reported in mammals [
175
]. Therefore, the optimum level
of NO could be different during normal and stress conditions. As a higher amount of
NO is formed through the reductive pathways during the O
2
limitation condition, it
would be beneficial that some amount of NO is scavenged from cells. For example, the
scavenging of NO using NO scavengers during hypoxia preserves the function of mammals’
mitochondria [
176
]. There may be several pathways of NO scavenging mechanisms during
O
2
-limited conditions, such as NO reduction to N
2
O [
24
,
177
] and the phytoglobin-NO
cycle in plants [89].
6.1. Nitric Oxide Reduction to Nitrous Oxide
We found very little information available on the role of N
2
O in plants (Table 3), which
could be due to the fact that N
2
O is less reactive to a biological system and is readily emitted
to the atmosphere. NO formation in plants is always suspected to be underestimated [
16
],
which could be due to that fact that NO is not simultaneously measured with N2O. Nitric
oxide can inhibit the activity of CcO either reversibly or irreversibly, such that a lower level
of NO can reversibly inhibit respiration, while a higher level of NO irreversibly inhibits it
due to RNS formation [
26
]. This reversible and irreversible inhibition of CcO could be linked
to NO reduction to N
2
O, as N
2
O is also involved in the reversible and partial inhibition of
Int. J. Mol. Sci. 2022,23, 11522 12 of 24
respiration at the site of CcO [
178
,
179
]. Moreover, CcO is known to reduce NO to N
2
O when
both NO and O
2
levels are low, while a higher level of NO can inhibit the NO reduction
process [
180
]. N
2
O is a relatively inert gaseous molecule, and its formation and release to
the atmosphere could significantly reduce the accumulation of RNS, while the inhibition of
NO reduction to N
2
O could increase the level of RNS that could irreversibly inhibit CcO.
Therefore, the dose-dependent effects of NO donors could be linked to NO reduction to
N
2
O, as the optimum NO level could favor N
2
O formation [
180
], while at a higher dose
of NO donor, NO could be high, thus favoring peroxynitirte formation, and thus exerting
negative effects. The use of tungsten as an NR inhibitor was reported to inhibit N
2
O
formation in plants [
181
], while NR inhibition challenged the plants’ survival, as described
in the above section, which further supports the concept that N
2
O formation also could
play a role in plants’ survival strategies. Moreover, recent results suggest that NR plays
critical role in NO-mediated N
2
O formation in microalgae Chlamydomonas reinhardtii [
182
].
Both NO [
183
] and N
2
O [
184
] can increase the activities of phenylalanine ammonialyase,
cinnamate-4-hydroxylase, and 4-coumaroyl-CoA ligase during pathogen attack in plants
while increasing total phenolic, flavonoid, and lignin content. Similarly, both NO and
N
2
O are reported to slow down fruit ripening by lowering ethylene synthesis during
post-harvest storage [
185
,
186
], while the role of NO depends on the optimum dose [
186
],
suggesting that NO could be reduced to N
2
O at the optimum dose, as discussed earlier.
Therefore, the similar roles of both NO and N
2
O observed in plants could be due to NO
reduction to N
2
O, which need further research as, to date, there is no research measuring
both NO and N
2
O simultaneously. Interestingly, not only during O
2
limitation [
187
,
188
]
but also during UV stress, plants are reported to emit more N
2
O [
189
], suggesting this NO
reduction to N
2
O could operate during other stresses too. Moreover, the intact chloroplast
of wheat was reported to produce N
2
O when supplied with NO
2−
[
190
], which could
be due to the possible reduction of NO
2−
to NO and NO to N
2
O, thus reducing the
toxicity of NO and protecting the chloroplast components as in mitochondria. A field study
showed a positive relationship between plant N
2
O emissions and photosynthetically active
radiation [
191
], supporting the concept of N
2
O production in chloroplasts, too. However,
to date, enzymes involved in NO reduction to N
2
O in the chloroplast are not clear, which
need further research.
Table 3. Beneficial activities of N2O in plants.
Beneficial Activities of N2O Reference
Using post-harvest technology, the storage of fruits under N2O can lower ethylene production and
slow the ripening of fruits. [185,192]
N2O can increase resistance to pathogens by improving the accumulation of total phenolic,
flavonoids, and lignin, as well as increase the activities of key enzymes in the metabolism of
phenylpropanol.
[184]
Can inhibit the browning activities of enzymes such as polyphenol oxidase (PPO) and/or peroxidase
(POD) and delay browning in fruits. [193]
Can delay decay, lower the respiratory rate, and maintain the quality of fruit. [194]
Microbial denitrification affects the pH by the production of OH
−
ions [
195
]. Moreover,
NO
3−
and NO
2−
supplementation in plants during hypoxia and anoxia has been reported
to improve cytoplasmic acidification as well as reduce the content of ethanol, which could
be toxic if accumulated in higher amounts, as reported in previous sections. The mechanism
behind the reduction in ethanol and lactate content and improved cytoplasmic acidification
by NO
3−
and NO
2−
could be due to ethanol and lactate acting as electron donors during
denitrification in plants and the release of OH
−
ions during the proposed denitrification
process in plants, as shown in Equation (1) [195].
5C2H5OH + 12NO3
−→6N2+ 10CO2+ 9H2O + 12OH (1)
Int. J. Mol. Sci. 2022,23, 11522 13 of 24
Many field-based studies have reported a positive relationship between plant N
2
O
emission and respiration rate [
187
,
191
]. This positive relation could be explained by Equa-
tion (1), as denitrification (N
2
O or N
2
or both) with a carbon source could release CO
2
,
resulting in the observed positive relationship between N
2
O emissions and respiration rate
in plants. A similar observation has been reported in microbial denitrification between
N
2
O and CO
2
emissions [
196
]. Nitric oxide reductase (Nor) in denitrifying bacteria uses
NADH as a reductase, while N
2
O is an intermediate [
70
]. Similarly, in the case of plant
mitochondria, the addition of NADH during hypoxia can increase the NO scavenging
rate [
197
], suggesting NO is reduced to N
2
O in a similar way to bacterial denitrification.
As N
2
O is a potent greenhouse gas that contributes to global warming and ozone deple-
tion [
177
,
198
–
201
], understanding its formation in plants is essential. Moreover, there exist
uncertainties in estimating the global N
2
O budget [
187
], which could be due to the fact that
sources of N
2
O are not well recognized [
24
,
187
]. For example, mainly soil-microorganisms
and fungi are considered as its natural sources [
198
], while plants are considered as a
medium to transport soil-microorganisms that produce N
2
O [
202
]. However, in a natural
environment, plant roots may face O
2
deficiency which favors reductive pathways of NO
formation along with NO reduction to N
2
O. This is supported by the fact that plants are
natural sources of both NO [
203
] and N
2
O [
187
] in field conditions too. Moreover, the
excessive use of N fertilizer along with waterlogging caused by heavy rainfall events under
the climate change scenario could trigger both NO and N
2
O production in plants, thus
increasing their atmospheric concentration.
6.2. Phytoglobin-NO Cycle
As stated in the above sections, the expression of Pgbs is beneficial for plants during
O
2
-limited conditions, which is due to the NO scavenging mechanism. For example, during
the germination of barley seeds, the scavenging of NO through the overexpression of Pgbs
resulted in a higher germination rate, protein content, and ATP/ADP ratios and a lower rate
of fermentation, the S-nitrosylation of proteins and S-nitrosoglutathione (GSNO) [
89
,
204
].
These lower levels of fermentation, S-nitrosylation of protein, and GSNO level in the
phytoglobin-overexpressing line could indicate that NO scavenging through Pgbs results
in lower stress as it is a marker of stress. However, NO scavenged through Pgbs could
be partially operated. In a low NO concentration, NO
3−
formed in a similar amount to
NO, while in a high NO concentration, there was no increase in NO
3−
[
205
], suggesting
that the phytoglobin-NO cycle could be a limiting factor for a higher level of NO formed
during O
2
deficiency. This is further supported by the fact that during hypoxia, about 80%
of NO is scavenged by mitochondria itself [
206
], while Pgbs are present in the cytoplasm,
chloroplast, and nucleus [
207
]. In this case, the recently proposed denitrification ability
in plants could be another mechanism of NO scavenging [
24
], as laboratory as well as
field-based studies have reported a significant amount of N
2
O formation in plants [
187
,
208
].
However, the proposed denitrification mechanism would also be a limiting factor during
anoxia or during high NO production conditions [
180
]. Another topic of interest would be
that the NO scavenging mechanisms would operate simultaneously or one after another so
that plants would benefit more from the mechanisms during O
2
deficiency. For example,
the scavenging of NO through Pgbs can reduce S-nitrosylation in plants [
89
]. Phytoglobin
expression in algae has been involved in the synthesis of not only NO but also N
2
O [
209
].
Although there is no direct evidence that Pgbs could reduce NO to N
2
O, it could play a
role in the phytoglobin-NO respiration cycle. The role of Pgbs could be due to its role of
oxidizing NO to NO
3−
during O
2
-limitation conditions, as NO
3−
is the precursor of N
2
O
formation in plants [
24
]. Moreover, phytoglobin-overexpressing mutants could maintain
a lower level of NO that can facilitate NO reduction to N
2
O during hypoxia. When NO
is reduced to N
2
O, N
2
O is released to the atmosphere, suggesting a beneficial way of
scavenging NO from the plant system, although N is lost from plants.
Int. J. Mol. Sci. 2022,23, 11522 14 of 24
7. Nitric-Oxide-Mediated Post-Translational Modifications and Their Roles during
Hypoxia and Anoxia
Recent research suggests that NO-mediated post-translational modifications are less
reported in plants during O
2
-limitation conditions [
210
], which could be due to the fact
that NO is reduced to N
2
O and emitted to the atmosphere. This could be supported by the
fact that plants emit very high N
2
O levels during waterlogging conditions, which are even
more than those in soil [
202
], while it has been recently suggested that the N
2
O emitted
from plants even in field conditions is produced in plant cells through NO reduction [
187
].
However, during complete anoxic conditions, this NO reduction to N
2
O would be in-
hibited [
180
]. Moreover, the NO-scavenging capacity of Pgbs would not operate during
complete anoxia [
211
], while they contribute to produce NO [
212
], thus, again, increasing
the level of NO, and thus promoting nitro-oxidative stress and inducing NO-mediated post-
translational modification in plants (Figure 2). Some of the NO-mediated post-translational
modifications and their roles in plants during O
2
-limited conditions are discussed in the
section below.
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 14 of 24
in plants [89]. Phytoglobin expression in algae has been involved in the synthesis of not
only NO but also N2O [209]. Although there is no direct evidence that Pgbs could reduce
NO to N2O, it could play a role in the phytoglobin-NO respiration cycle. The role of Pgbs
could be due to its role of oxidizing NO to NO3− during O2-limitation conditions, as NO3−
is the precursor of N2O formation in plants [24]. Moreover, phytoglobin-overexpressing
mutants could maintain a lower level of NO that can facilitate NO reduction to N2O dur-
ing hypoxia. When NO is reduced to N2O, N2O is released to the atmosphere, suggesting
a beneficial way of scavenging NO from the plant system, although N is lost from plants.
7. Nitric-Oxide-Mediated Post-Translational Modifications and Their Roles during
Hypoxia and Anoxia
Recent research suggests that NO-mediated post-translational modifications are less
reported in plants during O2-limitation conditions [210], which could be due to the fact
that NO is reduced to N2O and emitted to the atmosphere. This could be supported by the
fact that plants emit very high N2O levels during waterlogging conditions, which are even
more than those in soil [202], while it has been recently suggested that the N2O emitted
from plants even in field conditions is produced in plant cells through NO reduction [187].
However, during complete anoxic conditions, this NO reduction to N2O would be inhib-
ited [180]. Moreover, the NO-scavenging capacity of Pgbs would not operate during com-
plete anoxia [211], while they contribute to produce NO [212], thus, again, increasing the
level of NO, and thus promoting nitro-oxidative stress and inducing NO-mediated post-
translational modification in plants (Figure 2). Some of the NO-mediated post-transla-
tional modifications and their roles in plants during O2-limited conditions are discussed
in the section below.
Figure 2. Proposed model on mechanisms of hypoxia and anoxia tolerance as well as cell death by
NO3-NO2-NO pathway. The red arrows represent negative effects, while the green ones represent
positive effects.
Figure 2.
Proposed model on mechanisms of hypoxia and anoxia tolerance as well as cell death by
NO
3
-NO
2
-NO pathway. The red arrows represent negative effects, while the green ones represent
positive effects.
7.1. Protein Tyrosine Nitration (PTN)
Tyrosine nitration is the addition of a nitro group at the orthro position of the phenolic
hydroxyl group of tyrosine producing 3-nitrotrysoine [
213
,
214
]. OONO
−
can react with
CO
2
to give peroxynitous acid or ONOOCO
2−
which decomposes to carbonate radical
and nitrogen dioxide (NO
2
) [
215
], which could be a major pathway of tyrosine nitration
during O
2
deficiency, rather than being NO
2
- and O
2
-mediated. Several plant proteins
are a target of tyrosine nitration, which mostly decreases their activities [
215
,
216
]. In root
nodules, higher NO production was correlated with the PTN of glutamine synthetase, thus
decreasing its activity [
217
]. As PTN is a marker of nitrosative stress, we could conclude
that effective NO scavenging through Pgbs and N
2
O formation pathways would reduce the
PTN, thus enhancing the plants’ survival. For example, a plant inoculated with a bacterial
Int. J. Mol. Sci. 2022,23, 11522 15 of 24
strain that could detoxify the NO (flavohemoglobin) was reported to have the reduced
PTN of plant protein [217].
7.2. S-Nitrosylation
S-nitrosylation needs a preliminary reaction of NO with O
2
via the formation of higher
nitrogen oxides such as N
2
O
3
[
213
]. However, when the NO concentration is more than O
2−
,
the reaction between these two favors N
2
O
3
formation, thus leading to S-nitrosylation [
218
].
Hypoxia-related NO production is reported for protein’s S-nitrosylation [
219
]. Several plant
proteins are targets of S-nitrosylation and are inhibited [
213
,
220
,
221
], while some could lead
to an increase in resistance to oxidative stress [
221
]. Evidence suggests that Pgbs, catalase,
ascorbate peroxidase, and CcO are the targets of S-nitrosylation [
222
]. During hypoxia, NO
is involved in S-nitrosylation, targeting GSNO reductase for selective autophagy [
223
]. NO
can be converted to GSNO by GSH, while GSNOR converts GSNO to glutathione disulfide
(GSSG) and ammonia (NH
3
). Moreover, the induction of GSNOR enzyme under anoxic
conditions, more expressed in Pgb knockdown plants [
89
], can be simultaneously linked
to these NO-scavenging mechanisms and would depend on the NO concentration, as the
level of nitrosylation is only partially controlled by Pgb and GSNO reductase [
89
]. NO
can deplete GSH content in a dose-dependent manner in biological systems [
162
], thus
reducing the antioxidants’ availability. Moreover, peroxiredoxins (Prx), which play an
important role in combating ROS and ONOO−reductase activity, were inhibited through
S-nitrosylation in plants exposed to high NO which was produced during different stress
conditions [
31
]. As GSNO is a physiological NO donor, it also triggers nitrosative stress in
higher concentrations [224], thus increasing damage in the plant system.
7.3. Metal Nitrosylation
Nitric oxide forms metal-containing proteins by binding with the metal centers of
metalloprotein, known as metal nitrosylation [
225
]. The formation of the metal–nitrosyl
complex through the metal nitrosylation process can induce conformational changes in
the target proteins, impacting their activity [
226
]. Metal nitrosylation can prevent ROS
production by blocking the peroxidation of metals [
221
]. However, in the plant system,
hemoglobin, CcO, catalase, ascorbate peroxidase, and cytosolic and mitochondrial aconi-
tases are reported to be inhibited through metal nitrosylation [
227
], thus, again, affecting
plants. Therefore, an optimum level of NO is critical in reducing the negative effects.
8. Conclusions and Future Perspectives
All of this evidence suggests that the reductive pathways of NO formation are highly
beneficial, while these pathways are triggered during O
2
limitation, which could also
lead to NO toxicity. Therefore, effective NO scavenging mechanisms could help plants
survive for a longer duration during O
2
deficiency. However, considering NO scavenging
mechanisms during the O
2
-limitation conditions are defense mechanisms, the higher the
stress of O
2
limitation, the more nitro-oxidative stress there is in plants. So, we could
conclude that if this reductive pathway of NO formation and scavenging is finely tuned,
plants could be benefited in numerous ways. However, major scavenging systems could
be limiting factors which could explain why more NO production through the reductive
pathways leads to plant death in the model hypothesized in Figure 2. Although there has
been much advancement in understanding the reductive pathways of NO formation, we
suggest that scavenging mechanisms such as NO reduction to N
2
O in plants and their role
in reducing NO toxicity is not clear. The denitrification process, which is a major pathway
of nitrogen cycling in an ecosystem, is considered to be present only in micro-organisms
and some fungi, while we suggest that in plants, denitrification is involved in reducing NO
toxicity, thus enhancing plants’ survival in hypoxic and anoxic environments. Therefore,
future research could focus on the reductive pathways of NO along with N
2
O formation
while exploring the role of N
2
O formation in reducing nitro-oxidative stress. Moreover,
possible sites of N
2
O formation in plant cells need further investigation. Evidence suggests
Int. J. Mol. Sci. 2022,23, 11522 16 of 24
that not only during O
2
limitation conditions but also during other stresses, plants could
emit more N
2
O through the reductive pathways of NO formation, which needs further
research. As N
2
O is a potent greenhouse gas, understanding its formation in plants would
also help in addressing the current uncertainties in its global budget and implementing
mitigation strategies for its global warming effects.
Author Contributions:
Conceptualization, A.T., B.L. and C.H.; writing the draft manuscript, A.T.;
reviewing and editing the manuscript, W.D., M.H., B.L. and C.H.; supervision, B.L. and C.H. All
authors have read and agreed to the published version of the manuscript.
Funding:
This research was supported by funding from the National Key Research and Development
Program of China, (2021YFD1901104) and (2021YFD1700901), and the Key R&D Program of Hebei
Province (No. 21323601D, 19227312D).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
A.T. is thankful to China Postdoctoral Science Foundation for support. We are
thankful to Fiston Bizimana for his critical reading and comments on the draft manuscript.
Conflicts of Interest:
We declare that we do not have competing financial interests or personal
relationships that could have influenced this work.
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