2. 1. Introduction
Nitric oxide (NO) is now established as an important signalling
molecule regulating a wide range of physiological, biochemical and
molecular processes in animals [1]. The pathway of NO synthesis in
animals, mediated by isoforms of the enzyme nitric oxide synthase
(NOS) from L-arginine, is well characterized. In animals, NO plays a
vital role in maintaining blood pressure, host immune system, neu-
ral transmission and gene expression [2]. Nitric oxide is a gaseous
free radical molecule which diffuses readily through biological
membranes and has a biological half-life ranging from 5 to 13 s
[3]. This short half-life reflects the highly reactive nature of the
molecule: it reacts with metal complexes and other radicals, and
with biomolecules such as nucleic acids, proteins and lipids [4].
It can also react with superoxide (O2
À
) to form peroxynitrite
(ONOOÀ
), which can damage lipids, proteins and nucleic acids
[5]. There is growing evidence that NO is also an important signal-
ling molecule in plants [2,6–11] which has been obtained via the
application of NO, usually in the form of donors, via the measure-
ment of endogenous NO, and through the manipulation of content
by chemical and genetic means [12]. As in animals, the roles of NO
in plants may be equally diverse by having a role in cell signalling
and regulation of plant growth and development [6–10,13]. These
effects can be manifested directly by exogenous NO donors or
through effector molecules that preferentially regulate the redox
state of the cell [14]. The latter process occurs by the endogenous
generation of NO in plants either by metabolic regulations, pertur-
bations or modulations by developmental, environmental or
genetic cues [6–9]. NO can modify the structure and function of
haemoproteins or proteins by reversible iron-ligand binding to
functional haem prosthetic groups or to thiol groups and its role
as a radical cytotoxin or cytoprotectant through the redox homeo-
stasis of the cellular environment in plants has also been reported
[15–17]. These diverse effects of NO arise from concentration-
dependent reactions in living cells and tissue: low (sub-lmolar
concentrations) of exogenous NO donors stimulate plant growth,
regulation of stomatal movement and retardation of programmed
cell death and senescence [6–9,16,18–21] while higher (mmolar
concentrations) disturb metabolic activities in plant cells including
decreased photosynthetic electron transport, increased viscosity of
isolated thylakoid membrane lipid monolayers, leaf expansion
[18,22] and net photosynthesis [23].
Chloroplasts are very sensitive and susceptible to stressful cli-
matic changes [24–26] and because NO is a signalling molecule
in plants known to ameliorate stress effects, understanding its role
on chloroplasts is crucial. In addition, chloroplasts are proposed as
a site for the synthesis of endogenous nitric oxide [27–29]. Cur-
rently, despite many recent reviews on NO effects in plants [6–
9,14,30–35], little is known of the effect of NO on the regulation
of different physiological, biochemical and molecular processes in
chloroplasts. This review summarizes the most recent studies on
the effect of NO on chloroplasts with particular emphasis on the
effect of NO on the efficiency of the photochemical-transduction-
and carbon-fixation – pathways as well as its stress ameliorating
effect on chloroplasts.
2. Chloroplasts generate NO
Chloroplasts are semi-autonomous organelles in green plants
with diverse structure, function and environmental adaptability.
They have developed a thylakoid membrane network, suspended
in the stroma matrix: these membranes possess pigment protein
complexes which transduce solar energy by converting it to electri-
cal potentials that generate chemical energy (ATP) and reducing
equivalents (protons) in the form of NADPH+
generated by the
photolysis of water. The ATP and reductants generated are utilized
to reduce carbon dioxide to carbohydrates in the stroma.
The thylakoid membranes have four multi-subunit protein
complexes: photosystem II (PSII), photosystem I (PSI), cytochrome
b6f (cyt b6f) and ATP-synthase [36]. PSII and PSI are intrinsic pig-
ment–protein complexes spatially separated in the stacked (grana)
and unstacked (stromal lamellae) regions of the thylakoid mem-
branes in chloroplasts. PSII catalyzes the oxidation of water and
the reduction of plastoquinone [37,38]. Cyt b6f complex accepts
an electron from plastoquinol and donates it to PSI via plastocya-
nin. The electron is transferred ultimately to the NADPH+
via PSI,
and then released to the stroma to be utilized in the CO2 reduction
process. Concomitantly with the electron transfer, the accumulated
protons in the lumen are used for ATP synthesis by ATPase.
Production of NO in plant cells arises from several different
pathways and in different organelles [30,39,40] including mito-
chondria [40], peroxisomes [41] and the chloroplast [27,28].
Dependant on concentration, NO can provoke both beneficial and
harmful effects which also depend on its location within the plant
cell [42,43]. In plants, NO can be produced from nitrite [44], from L-
arginine by NOS-like biochemical pathways [41] and from S-nitro-
soglutathione by decomposition [45]. Endogenous NO generation
in chloroplasts without any external donors has been recently
reported [27,28]. It is notable that NO synthesis was first detected
in chloroplasts in response to various stressors like high tempera-
ture, salinity, iron, elicitors or osmotic stress, using NO-sensitive
diaminofluorescein probes [46–48].
Nitric oxide synthase (NOS) activity was first detected in fungi
and a higher plant by Ninnemann and Maier [49]. Despite evidence
of an Arg-dependent pathway for NO synthesis in higher plants, no
NOS homologs have been identified in plants [50]. Guo et al. [51]
described the identification of a plant NOS gene involved in hor-
monal signalling (Atnos1); however, since no NOS activity was
detected in the purified AtNOS1 protein [52,53], it was renamed
AtNOA1 (nitric oxide associated 1), because it appeared essential
for NO generation in the cell. Another enzymatic source of NO in
plants is the cytosolic nitrate reductase (NR) [12] and the NO so
produced can readily diffuse into the chloroplast stroma [43].
Nitrite can also be reduced to NO non-enzymatically at acidic pH
values [54]. Non-enzymatic, light-mediated conversion of nitrogen
dioxide to nitric oxide by the plastid carotenoids has also been doc-
umented [55].
3. Target sites of NO action in chloroplasts
The effect of NO on target sites in the chloroplast and on its pho-
tosynthetic apparatus has mostly been accumulated by applying
exogenous gaseous NO or by various NO donors on leaves, leaf
discs, leaf extracts, intact chloroplasts or isolated chloroplast com-
ponents. The results are often confusing and contradictory but, in
this review, an effort is made to clarify such discrepancies.
3.1. Effects of NO on photosystem II
3.1.1. Electron transport chain
Several electron transport chain components of PSII have been
identified as target sites for NO in chloroplasts. EPR and chloro-
phyll fluorescence analysis of isolated PSII-enriched membranes
and chloroplasts treated with NO gas demonstrated NO binding
to the non-haem iron of PSII between QA and QB quinone binding
sites, namely, QAFe2+
QB [56,57], at the catalytic Mn cluster of the
oxygen evolving complex (OEC) [58] and at the YD tyrosine residue
of D2 protein [59]. The proposed target sites of NO binding are
illustrated in Fig. 1.
36 A.N. Misra et al. / Nitric Oxide 39 (2014) 35–45
3. The QAFe2+
QB binding site of NO (Fig. 1) is also the bicarbonate
binding site on the acceptor side of PSII, which is detailed studied
and characterized binding site of NO in chloroplasts [56,57]. Diner
and Petrouleas [57] first established that the NO-binding leads to a
significant (10-fold) decrease of the electron transfer rate between
QA and QB [57]. This effect is reversible by the addition of bicarbon-
ate [57]. Binding of NO to the QAFe2+
QB complex is facilitated in the
presence of reduced primary electron acceptor QA
À
, as this
reduction weakens the bond between bicarbonate and iron [60].
In this process, NO could act as an anion in displacing bicarbonate
from its binding site [61]. Wodala et al. [62–64], using pea leaf
discs treated with NO released from sodiumnitroprusside (SNP),
S-nitroso-N-acetylpenicillinamine (SNAP) and S-nitrosoglutathi-
one (GSNO), confirmed the decreased electron transport rate
between QA and QB [57] and thus, also demonstrated, in vivo, that
NO causes increased time constants for the QA
À
dark reoxidation
and inhibited charge recombination of QA
À
with the S2 state of the
OEC.
Similar results have been found, both in vitro and in vivo, with
higher concentration of exogenous NO suggesting significant donor
and acceptor side inhibition of electron transport. However, our
PAM fluorescence and flash oxygen evolution studies on SNP treated
isolated pea thylakoid membranes, demonstrated that the electron
donor side of PSII is the probable target site of NO action [65]. In this
study we have clearly shown that these effects are due to the NO
released by SNP [65] and not to the accompanying CNÀ
as previously
reported for SNP treated leaves [62,63]. Comparison of the effects of
SNP [65] with other NO donors (SNAP and NOR2 (±)-(E)-4-methyl-2-
[(E)-hydroxyimino]-5-nitro-3-hexenamide) on isolated thylakoid
membranes reveals that only SNP stimulates the overall electron
transport flux through the PSII. These results indicate either
NO-donor specificity for the NO target sites in PSII and/or a depen-
dence on the concentration of the released NO. Thus, very low NO
concentrations below $70 nM, for which we observed maximum
stimulation of PSII ETR [65] should be tested.
3.1.2. Effects of NO on PSII photochemistry
The PAM chlorophyll fluorescence is a powerful method for
characterizing the photochemical and non-photochemical energy
conversion properties of PSII (for reviews see [66–69]) and has
been used to quantify the effects of NO on such properties, namely,
the maximal quantum efficiency of PSII photochemistry in dark
(Fv/Fm) and light (Fv0
/Fm0
) adapted states, the photochemical
quenching coefficient (qP), the effective quantum yield (efficiency)
of PSII photochemistry in light-adapted state (UPSII = qP Á Fv0
/Fm0
),
the electron transport rate (ETR, which is proportional to UPSII)
and the non-photochemical quenching (NPQ). The different NO
donors, SNP, GSNO and SNAP produce contradictory results on
these parameters for leaves and isolated thylakoid membranes
under non-stress conditions (Table 1) or under stressful environ-
mental conditions (Table 2). In leaves under non-stress conditions,
NO derived from SNP and GSNO decreases the maximum quantum
efficiency (Fv/Fm), indicating an inhibitory effect on PSII photo-
chemistry [62,63,70]. In contrast, SNP treated seedlings during
greening [71] or SNP-treated leaves under stress by heat, salt, Fe
deficiency, metal toxicity, UVB and drought conditions exhibit
higher Fv/Fm when compared with the respective stressed control
leaves not treated with SNP (Table 2), thus demonstrating the
stimulating effect of SNP produced NO on PSII photochemistry
which is correlated with an increase in the proportion of the open
PSII reaction centres. With some exceptions, all of the NO donors
applied to isolated thylakoid membranes [72] or leaves
[62,63,70] induced a decrease in effective quantum yield UPSII
which is related to photochemical quenching (qP) and the ETR:
however, the exceptions included SNP on isolated thylakoid
membranes [65] or in vivo during greening [71] and stress condi-
tions [73,74] and SNAP on leaf disks [62] (cf. Table 1, Table 2).
Noa1-suppressed mutants [75] exhibit lower PSII efficiency
(Fv/Fm, UPSII) in comparison with the wild type (WT), suggesting
a decrease in the energy transfer efficiency within PSII in the
mutant seedlings deficient in endogenous NO production (Table 3).
Similar to the wild type plants under stress (Table 2), exogenous
NO donated by 50 lM SNP to Atnos1 mutant plants ameliorated
the methyl viologen induced decrease in the photochemical
efficiency (Fv/Fm) (Table 3).
NO donated by GSNO in vivo, in pea leaf disks, decreases steady-
state non-photochemical quenching (NPQ) and energy-dependent
quenching (qE) in a concentration-dependent manner [62] in WT
pea leaves. SNP released NO in vitro, in isolated pea thylakoid
membranes, also diminishes the NPQ which demonstrates the
decreased excitation energy dissipation as heat resulting from
NO induced structural reorganizations in PSII and its antenna com-
plexes [65]. Recently, Ördög et al. [76] demonstrated that chloro-
plasts from stomatal guard cells, when exposed for very short
time (<10 min) to exogenous NO (450 nM) derived from GSNO,
responded by instantly decreasing photochemical fluorescence
quenching coefficients (qP and qL), the effective quantum yield
(efficiency) of photosystem II (UPSII) and non-photochemical
quenching (NPQ) values close to zero. This effect of NO in vivo is
reversible since the electron transport rate (ETR) could be restored
by bicarbonate [76] which is known to compete with NO binding
to QAFe2+
QB complex [60].
Wodala et al. [62] suggested that the different chemical proper-
ties of NO donors and different experimental conditions generated
conflicting experimental results in vivo and Ördög et al. [76] pro-
posed that they may be explained by the different NO donor and/
or biological samples used in the experiments. However, applica-
tion of the same NO donor (SNP) suggests that NO concentration
and exposure time is also important for observing the effect of
NO on PSII photochemistry involving QA
À
reoxidation, electron
transport rate, photochemical and nonphotochemical quenching
in vitro (see Table 1, Ref. [65]). Importantly, we have also shown
that the concentration dependence of the stimulating effect of
SNP donated NO is biphasic; there is a maximum at about 70 nM
NO (25 lM SNP) and then a decrease at higher concentrations [65].
3.1.3. Oxygen evolving complex
Oxygen evolving complex (OEC) of PSII catalyses the oxidation
of water to molecular oxygen and comprises a four-manganese-
atom cluster with one Ca cation and at least one Cl anion (for
review, see [69]). The functional conformation of the Mn cluster
Fig. 1. Schematic presentation of the proposed target sites of NO in the PSII
complex.
A.N. Misra et al. / Nitric Oxide 39 (2014) 35–45 37
4. is thought to be maintained by a 33 kDa hydrophilic protein sub-
unit of OEC attached to the luminal side of the D1/D2 heterodimer.
During the oxidation of two water molecules to one diatomic oxy-
gen molecule and four protons, the Mn cluster cycles through four
stable states (S0–S3) and one transient state (S4) (Fig. 2). Dark-
adapted photosynthetic apparatus contains mainly S0 (20–30%)
and S1 (80–70%) states, as S2 and S3 states deactivate back to S1.
The most reduced state is S0, while S1, S2 and S3 represent higher
Table 1
Exogenous NO-induced changes in photosynthetic parameters and protein complexes of chloroplasts under non-stress conditions.
Plant material NO donor, concentration, time of
exposure and [NO concentration]
NO effect Refs.
PSII membranes
[Spinach]
NO gas, [0.003–1.5 mM] Disappearance of EPR Signal II (arising from TyrD+
at the donor side of PSII) with a Kd of
3 lM NO
[56]
Formation of NO complex with the non-haem Fe(II) between QA and QB at the acceptor
side of PSII with a Kd of 250 lM NO
PSII membranes
[Spinach]
NO gas, [0.6 mM] Decreased flash-induced O2 yields [82]
NO gas, [0.5–0.7 mM], 3–5 h Decreased O2 evolution activity (65%) [81]
NO gas, [0.6–2 mM], 4–6 h Decreased flash-induced O2 yields [58]
Thylakoid membranes
[Pea]
SNP, 5–100 lM, 20 min in dark [65]
Up to 10 lM, [635 nM]*
Decreased NPQ (4Â), qN (2Â)
Up to 25 lM, [670 nM] Increased qP, UPSII, ETR ($2.6–2.7Â). Decreased flash-induced O2 yields
>25–100 lM, [>70–200 nM] Full inhibition of the O2 evolution
Thylakoid membranes
[Spinach]
NOR2, 400 lM, 1 min, [1 lM] Decreased rate of ATP synthesis. (SNAP and NOR2 releasing 1 lM NO have the same
effect)
[72]
SNAP, 1 min, [1–10 lM] Decreased rate of ATP synthesis (IC50 = 0.7 lM NO), DpH formation across the
membrane (IC50 = 2 lM NO), ETR (IC50 = 3 lM NO) and ATPase (ATP hydrolysis)
activity (IC50 = 50 lM NO)
Isolated chloroplasts
[Soybean]
GSNO, 50–500 lM, 30 min, [up to 3 lM] Decreased O2 evolution rate (18%) [29]
Isolated chloroplasts
[Spinach]
NO gas, [30 lM], 45 min Decreased electron transfer rate between QA and QB (10Â) [57]
NO gas, [0.003–0.5 mM] Disappearance of EPR Signal II (from TyrD + at the donor site of PSII, Kd = 3 lM NO).
Formation of NO complex with the non-haem Fe(II) between QA and QB at the acceptor
side of PSII (Kd = 30 lM NO)
[56]
Isolated chloroplasts
[Wheat]
SNP, 0.05–0.2 mM , 12 h dark Alleviation of Rubisco degradation [91]
SNP, 1–5 mM, 12 h dark Enhanced Rubisco degradation
Guard cells of leaf
epidermis [broad
bean]
GSNO, 50 lM, fast perfusion, [450 nM] Decreased Fv/Fm, qP, UPSII, ETR, qL, and NPQ. Increased Fv0
/Fm0
[76]
Cell suspension
cultures
[Arabidopsis]
SNP, 250 lM, 20 min Proteins S-nitrosylated (nitrosothiol content of $2 lmoles/mg protein) [93]
GSNO, 250 lM, 20 min Proteins S-nitrosylated (nitrosothiol content of $40 lmoles/mg protein)
Greening etiolated
seedling [Barley]
SNP, 100 lM, 1 h vaccum infiltrated.
Analysed for 36 h
Increased Fv/Fm and UPSII at 12 h and 24 h, compared to seedlings without SNP [71]
Leaf [Tomato seedlings] SNP, 100 lM in nutrient, 8 d Increased qP, ETR and Fv0
/Fm0
. Decreased qN [138]
Leaf [Wheat seedlings] SNP, 100 lM via watering every day, 8 h/
d light, 8 d
Increased Fv/Fm and ATPase activity [90]
Excised leaf [Potato] SNP via petioles, P 150 lM, 8 h + light Decreased Fv/Fm and UPSII with an increase in SNP concentration [70]
SNP via petioles, 150 lM, 4–12 h + light Decreased qP and UPSII ($20%)
Leaf discs [Potato] SNP, 150 lM, 4–12 h + light Decreased UPSII (2.7Â – 3Â)
Leaf [Medicago
truncatula]
SNP, 2.5 mM via vacuum infiltration, 3 or
24 h
Decreased Fv/Fm and photosynthetic rate [139]
Leaf [Wheat seedlings] SNP, 0.05–0.1 mM, spray every 3d Alleviation of Rubisco degradation [91]
SNP, 0.5 mM, spray every 3d Enhanced Rubisco degradation
Leaf discs [Pea] SNP, 200 lM, 2 h light, [0.156 lM]**
Decreased Fv/Fm, qP, UPSII, and ETR. Increased NPQ [63]
SNAP, 1 mM, 2 h, light, [0.48 lM] QA
À
reoxidation kinetics and chlorophyll fluorescence parameters not significantly
changed
[62]
SNP, 0.2–1 mM, 2 h light Decreased Fv/Fm and ETR with an increase of SNP concentration [64]
[60.78 lM] Increased NPQ and in a certain high SNP range decreased
[0.78 lM] Increased time constants for QA
À
dark reoxidation (fast and middle phase)
GSNO, 1 mM 70 min, light Increased PSI quantum efficiency, pool sizes of electrons in the intersystem chain and
from stromal donors
[85]
GSNO, 1 mM 2 h, light, [2.70 lM] Decreased Fv/Fm, qP, UPSII, NPQ. Electron transfer rate between QA and QB (the time
constant of the middle phase of QA
À
reoxidation increased (2Â)). Inhibited charge
recombination of QA
À
and S2 state of the OEC
[62,63]
Leaf discs and leaf
exctract [Kalanchoe
pinnata]
GSNO, 25–500 lM, in leaf exctract,
20 min dark or leaf disc with 250 lM, 2 h
dark
Both large and small subunits of Rubisco (Rubisco S and Rubisco L) S-nitrosylated.
Inhibition of Rubisco activity
[92]
Leaf [Mung bean] SNP, 0.5 mM, 6 h, light, [1 lM] Decreased quantity of Rubisco activase and the b-subunit of the Rubisco subunit-
binding protein ($5Â)
[84]
Increased amount of 33 kDa polypeptide of OEC ($5Â)
Plants [Spirodela
oligorrhiza]
SNOC or SIN-1 1 mM, 1 h, light Inhibition of D1 protein phosphorylation. Degradation of D1 not inhibited [140]
Leaf [Arabidopsis
thaliana]
NO gas, [1.25 mM], 10 min, light Proteins S-nitrosylated (nitrosothiol content $29 lmoles/mg protein) [93]
Leaf [Oat and alfalfa] NO gas fumigation, [2.5–9 ppm], 100 min Decreased apparent photosynthesis and CO2 uptake rate [23]
*
Extrapolated value based on the data given in [65].
**
Extrapolated value based on the data given in [62].
38 A.N. Misra et al. / Nitric Oxide 39 (2014) 35–45
5. Table 3
Summary of NO-mutants used to characterise effects of endogenous NO on photosynthetic characteristics and components.
NO-Mutant Endogenous NO production Mutant photosynthesis parameters and protein complexes in chloroplasts vs wild type (WT) Refs.
Arabidopsis thaliana
nos1 Decreased Decreased Fv/Fm rapidly than in WT after NaCl and methyl viologen (MV) treatments [128]
Exogenous application of 50 lM SNP increased Fv/Fm under MV treatment
noa1 Chlorophyll and Rubisco levels are slightly (at 22 °C) and strongly (at 12 °C) lowered as
compared to those in WT
[110]
nos1/noa1 Decreased Fv/Fm with the progression of dark-induced leaf senescence than in WT [112]
Rice (Oryza sativa)
noa1/rif1 Decreased Decreased Fv/Fm and UPSII ($50%) [75]
Down regulation of PSI, PSII, cyt b6f, ATP synthase, NADP reductase and Rubisco
noa1 Chlorophyll biosynthesis and Rubisco formation are suppressed at lower (22 °C) but not at
high (30 °C) temperature without significant change in Pn than in WT. Down regulation of the
expression of genes for RbcL, PsbC, PsbD, Fd-NADP+
reductase and ATPase e-subunit
[110]
Table 2
Exogenous NO-induced changes in photosynthetic parameters and protein complexes of chloroplasts in leaves under stress conditions.
Stress treatment Plant material NO-donor treatment NO effect compared with respective
stressed controls
Refs.
Heat stress [45 °C]
90 min, dark Leaf discs [Mung bean] Presoaking leaf discs in SNP solution (150 lM SNP,
60 min in low light)
Increased Fv/Fm (16.5%) [141]
3–24 h, normal light Leaf [Chrysanthemum] Pretreatment for 3 d with 200 lM SNP spray, twice
a day in the morning and evening
Alleviation of the decrease in Fv/Fm, UPSII
and Pn, and alleviation of an increase in
NPQ
[142]
NaCl salt stress
50 mM, 8 d Leaf [Cucumber
seedlings]
Combined (with the stress) treatment with 10–
400 lM SNP in nutrient medium under natural light
Increased Pn with maximal effect at 50
and 100 lM SNP
[143]
100 mM, 5 h Leaf [Rice seedlings] Pretreatment for 2 d with 1–1000 lM SNP in
hydroponic solution and subsequently 8 d grown
under NaCl stress conditions with SNP
Increased UPSII, maximal effect with 1 lM
SNP pretreatment
[144]
150 mM, 16 d Leaf [Sour orange plant] 48 h pretreatment with 100 lM SNP before salt
stress
Increased Pn [95]
1 mM, 8 d Leaf [Tomato seedlings] 100 lM SNP in nutrient solution with NaCl Alleviation of the decrease in Fv/Fm, Fv0
/
Fm0
, qP, ETR and Pn, and alleviation of an
increase in qN
[138]
Fe deficiency
50 lM Fe(III)-EDTA, 20 d Leaf [Maize plants] 20-d-old plants grown in nutrient medium with
100 lM SNP
Increased psbA and rbcL gene abundance
(75%)
[101]
0.1% (w/v) FeSO4 Leaf [Peanut plants] Plants grown till flowering phase treated with
1 mM SNP by spraying or through fertilizer
treatments
Increased Pn, Fv/Fm and UPSII [74]
Metal toxicity
1.2 mM AlCl3Á6H2O, 7 weeks Leaf [Sour pummelo
seedlings]
Plants grown in nutrient medium with 10 lM SNP Alleviation of the decrease in Fv/Fm, the
inactivation of OEC and the impairment of
whole chain (H2O-NADP) photosynthetic
electron transport
[145]
5 lM CdCl2, 25 d Barley seedlings Plants grown in nutrient medium with 250 lM SNP Increased Pn [146]
0.2 mM CdCl2, 10 d Rice (Oryza sativa)
plantlets
Plants grown in nutrient medium with 5–500 lM
SNP
Increased Pn at 100 lM SNP [147]
UV-B radiation
15 lmol mÀ2
sÀ1
for 0 to 96 h Leaf [Bean] 100 lM SNP, every 4 h co-treatment by spraying
the leaves
Increased Fv/Fm and UPSII [73]
6 W mÀ2
sÀ1
during the 14 h
light period, 3d
Leaf [Maize seedlings] 0.1 mM SNP, 3 d at 6-h intervals co-treatment by
spraying the leaf surfaces
Increased Fv/Fm [148]
10.08 kJ mÀ2
dÀ1
Leaf [Wheat seedlings] 100 lM SNP, 8 d, 8 h/d co-treatment by watering Increased Fv/Fm and ATPase activity [90]
Drought or osmotic stress
À0.5 MPa PEG Leaf [Wheat seedlings] 100 lM SNP, 3 d co-treatment with solution to the
soil
Increased qP and Fv0
/Fm0
[149]
15% PEG6000 0.3 mM SNP, 24 h co-treatment from nutrient
solution
Increased Fm/Fo, Fv/Fm and Pn [150]
Herbicides
Atrazine (100 lg/L) or
glufosinate (10 mg/L)
Green algal cells
[Chlorella vulgaris]
20 lM SNP in culture media for 48 h Up-regulated expression of psbC, psaB,
chlB, and rbcL genes
[100]
100 lM SNP in culture media for 48 h Down-regulated expression of psbC, psaB,
chlB, and rbcL genes
4 mg LÀ1
diquat, 2 h Isolated chloroplasts
[Potato]
SNP (100 lM) or SNAP (200 lM), 24 h pretreatment
by spraying leaves before herbicide treatment for
2 h in light and after that isolation of chloroplasts
Decreased electron transport through
both PSII and PSI
[99]
A.N. Misra et al. / Nitric Oxide 39 (2014) 35–45 39
6. oxidation states and molecular oxygen being evolved at the transi-
tion from S4 to S0 state [77,78].
It is well established that NO rapidly destabilizes the excited
states of the Mn cluster complex. Schansker et al. [58] studied
the effect of NO on oxygen oscillation patterns of PSII-enriched
membranes and observed a shift of the maximum flash-induced
oxygen yield from flash 3 to flash 6/7 possibly caused by NO reduc-
tion of the Mn cluster to the SÀ2 state which is represented by the
Mn(II)–Mn(III) dimer. The action of NO as a reductant was demon-
strated with EPR spectroscopy [58,79–81]. In our recent in vitro
study we demonstrated that the SNP (above 5 lM) inhibited the
primary oxygen-evolving reactions at the electron donor side of
the photosynthetic apparatus [65]. Similar inhibition of oxygen
evolution was reported in intact chloroplasts from soybean leaves
when treated with 500 lM GSNO ($3 lM NO) for 30 min [29].
These effects on the PSII donor side could arise by interaction of
NO with the YD tyrosine of D2 protein [59] forming a YD
Å
–NO couple
with a redox potential low enough to become a more efficient elec-
tron donor in isolated thylakoid membranes than the redox-active
YZ tyrosine, of the D1 protein.
The NO molecule can occupy the open coordination position,
forming a terminal Mn–NO complex. One-electron reduction of
the cluster can occur followed by release of NO2
À
(Mnn
+ NO ? Mnn
-
À1
+ NO+
, NO+
+ OHÀ
? NO2
À
+ H+
) as it was also proposed as a sin-
gle exception for interaction of NO with the S3 state [82]. This is a
fast two-electron reduction with five times higher rate than the
reduction rate of the S2 state (see Fig. 2) [82]. The rapid interaction
of NO with S states of the OEC is compatible with a metallo-radical
character of these states. Most probable role of Tyr YD
Å
in oxidizing
of Mn complex to the lower oxidizing state S0 than the S1 state is
explained for such an interaction of NO [83]. This observation cor-
relates with the observed increase of Mn population in the most
reduced S0 state [65]. Exogenous NO donor SNP increases the turn-
over time of the oxygen evolving centres, i.e. it delays the process of
the electron donation or Si states turnover, which could be the
result of structural reorganizations, which affect the interactions
between PSII and its antenna complexes after SNP treatment [65].
In addition, Lum et al. [84] showed that the treatment of mung bean
leaves with SNP increased the content of the 33 kDa protein of OEC,
which maintains the functional conformation of the Mn cluster.
Table 1 shows that all reports so far have demonstrated a
decrease of photosynthetic O2 evolution in a NO concentration
dependent manner. Our experiments even showed O2 consump-
tion above 25 lM SNP [65].
3.2. Effect of NO on the photosystem I activity
Wodala and Horváth [85] studied the effect of exogenous NO
from donor GSNO on PSI photochemistry by measuring P700
absorbance changes in intact pea leaves and revealed that NO
increases PSI quantum efficiency and the pool size of electrons in
the intersystem chain. Further experiments with lower (<70 nM)
NO concentrations, however, are required for a better understand-
ing of NO effect on PSI.
3.3. Interaction of NO with cytochrome b6f complex
The cytochrome b6f complex is the photosynthetic electron
transport mediator between PSII and PSI transferring electrons
from water to NADP+
. Compared with its analogue cyt bc1 (com-
plex III) in mitochondria and bacteria, the cyt b6f has one additional
redox cofactor, the haem cn [86,87] but its function is still not clear.
Recently, Twigg et al. [88], using EPR spectroscopy on isolated cyt
b6f, demonstrated that reduced (ferrous) haem cn binds NO both
gaseous or from NO donor, diethylamine NONOate: this is the first
example of haem iron of a chloroplast haemoprotein being a target
for NO binding [4]. Future work may determine if NO, by interac-
tion with haem cn of cyt b6f complex, can be involved in signalling
pathways by modulation of cyclic electron flow around PSI and so
regulate redox potential in chloroplasts, thereby inhibiting of pho-
tosynthetic electron transport in a manner similar to that of NO
interaction with mitochondrial cyt c oxidase which, in turn, affects
mitochondrial electron transport [89].
Fig. 2. The effect of NO on the classic Kok’s S-cycle model for oxygen evolution during illumination, involving four stable states (S0–S3) and one transient state (S4). It shows
the oxidation states of the four Mn ions in different S-states proposed by Hoganson and Babcock [77]. Dashed arrows indicate reduction of S-states in the darkness. The
arrows labeled with NO show the probable NO-mediated reduction of the S-states in darkness [58,81,82].
40 A.N. Misra et al. / Nitric Oxide 39 (2014) 35–45
7. 3.4. Photophosphorylation
Takahashi and Yamasaki [72] reported that the exogenous NO
donor, SNAP, not only strongly inhibited ATP formation by photo-
phosphorylation (IC50 = 0.7 lM NO) in isolated spinach thylakoid
membranes but also the inhibition was reversed by high bicarbon-
ate concentrations. Also inhibited, but to a lesser extent, was elec-
tron transport rate (ETR) (IC50 = 3 lM NO) and light-induced DpH
formation (IC50 = 2 lM NO) across thylakoid membrane while
higher concentrations (IC50 = 50 lM NO) inhibited ATP hydrolysis
[72]. Comparing IC50 values for ATP synthesis and hydrolysis, the
authors suggest that NO-induced inhibition of photophosphoryla-
tion cannot be attributed to inactivation of the H+
-ATPase [72].
Recently, however, Yang et al. [90] showed that treatment of
wheat (Triticum aestivum) seedling leaves with 100 lM SNP
enhanced ATPase activity but the NO concentration was not
reported. Based on NO released by 100 lM SNP (see Table 1, Ref.
[65]), it would be lower than the concentrations inducing
decreased ATPase activity [72] (Table 1) suggesting that NO can
enhance ATPase activity at low concentration and inhibit at higher
concentrations.
3.5. Modulation of Carbon dioxide reduction
In 1970 Hill and Bennett [23] first reported that CO2 assimila-
tion rates were inhibited by increased NO gas fumigation of trees.
Tu et al. [91] studied the NO concentration dependence of Rubisco
degradation both in vivo (wheat leaves) and in vitro (isolated wheat
chloroplasts). They showed two concentration-dependent effects
of NO on aging-induced degradation of Rubisco polypeptide
(Table 1). At low SNP concentrations below 0.1 mM for leaves
and 0.2 mM for chloroplasts Rubisco degradation is diminished
but is enhanced at higher SNP concentrations (0.5 mM for leaves
and 1 mM for chloroplasts). Lum et al. [84] reported that 0.5 mM
SNP (1 lM NO) under 6 h light treatment decreases the amount
of Rubisco activase and the b-subunit of the Rubisco subunit bind-
ing protein. Abat et al. [92] identified a number of soluble proteins
in chloroplast stroma of a CAM plant Kalanchoe pinnata, associated
with carbon (fructose-bisphosphate aldolase, glyceraldehyde 3-
phosphate dehydrogenase, phospho-glycerate kinase, UDP-glucose
4-epimerase), nitrogen (glutamate ammonia ligase) and sulphur
(cobalamine-independent methionine synthase) metabolism,
stress (putative HSP and HSP 81-3) and photosynthesis associated
proteins (e.g. Rubisco, phosphoenolpyruvate carboxylase, carbonic
anhydrase, glycolate oxidase, etc.), as targets of NO action via S-nit-
rosylation. They also observed that both the subunits of Rubisco
were S-nitrosylated and that Rubisco activity decreased in a NO-
dose-dependent manner [92]. Previously, Lindermayr et al. [93]
identified all the above enzymes associated with carbon metabo-
lism, except the putative UDP glucose 4-epimerase, as targets for
S-nitrosylation in Arabidopsis. The fact that enzymes in carbon,
nitrogen and sulphur metabolism are targets of S-nitrosylation
may have implications in the regulation of these pathways in
plants and perhaps especially in stress conditions [92]. Further-
more, NO regulates superoxide production by the inhibition of
NADPH oxidases via S-nitrosylation and also induces the activity
and expression of antioxidant enzymes [94]. Tanou et al. [95] dem-
onstrated that SNP pre-treatment (100 lM, 48 h) of Citrus plant
before salinity stress (150 mM, 16 d) alleviated salinity-induced
protein S-nitrosylation and reduces the levels of leaf S-nitrosylated
proteins to those of unstressed control plants. Their results indi-
cated also an overlap between H2O2- and NO-signalling pathways
in acclimation to salinity and suggested that the oxidation and S-
nitrosylation patterns of leaf proteins are specific molecular signa-
tures of citrus plant vigour under stressful conditions. Tanou et al.
[95] and other authors (Table 2) also reported an increase in the
net photosynthetic rate (Pn) of stressed plants treated with exoge-
nous NO.
4. Regulation of plastid gene expression
Genetic investigations of Arabidopsis mutants showed an impor-
tant role of NO in photosynthetic gene regulation [96–98]. Beligni
and Lamattina [99] found that 1 day pre-treatment of tomato
leaves with 100 lM SNP (or 200 lM SNAP) counteracted diquat-
mediated decrease in Rubisco (rbcLS) mRNA levels during the first
4 h of diquat treatment and delayed total RNA degradation after
longer (7–12 h) treatment. Herbicides, like diquat, cause total oxi-
dative degradation of RNA, while NO countered this by acting as an
antioxidant in vivo [99]. Total protein analysis of potato leaves pre-
treated for 1 day with 100 lM SNP or 200 lM SNAP and then
diquat treated for 36 h showed complete protection of the Rubisco
large subunit only indicating that in addition to direct reactive oxy-
gen species (ROS) scavenging by NO, other mechanisms such as call
signalling, could explain selective protection of the Rubisco large
subunit by NO [99].
Later, Qian et al. [100] showed that 48 h exposure to NO
donated from 20 lM SNP to either atrazine- or glufosinate-inhib-
ited green algae Chlorella vulgaris, up regulated photosynthesis-
related genes psbC (encoding the CP47 PSII core subunit), psaB
(encoding one of the two PSI core subunits), chlB (necessary for
the light-independent Chl synthesis) and rbcL (encoding the Rubi-
sco large subunit) expression to the control level [100] (Table 2).
The expression of these four genes is necessary for the photosyn-
thetic electron transport chain, synthesis of chlorophyll and carbon
fixation, respectively. However, higher concentration of SNP
(100 lM) had opposite effect – it decreased the expression of these
four genes [100]. Graziano et al. [101] studied Fe-deficient maize
plants co-treated with 100 lM SNP during growth and found that
the exogenous NO induced accumulation of transcripts encoding
both the D1 protein of PSII (psbA) and the Rubisco large subunit
(rbcL).
5. Effect of NO on photosynthesis under physiological and stress
conditions
In recent years, NO has been shown to be involved in many key
physiological processes in plants under normal and stress condi-
tions and that is involved in almost every stress response [6–9]
including the amelioration of certain specific stresses
[2,8,14,21,97,98] (Table 2). Many plants produce substantial
amounts of NO in their natural environments [102]. Prolonged
exposure to stress, however, may result in enhanced production
of NO and its derivatives, resulting in nitrosative stress (NO-stress)
[14]: the nitrosylation of lipids, proteins and nucleic acids leads to
severe metabolic impairment and degradation of cellular metabo-
lites leading to programmed cell death [8,9].
5.1. Development and senescence
Photosynthetic pigments, especially chlorophyll (Chl), are visible
markers for both chloroplast development and senescence in leaves
[103–108]. The synthesis of chlorophyll and plastid proteins is intri-
cately connected during chloroplast development and is essential
both for the stability of Chl–protein complexes in vivo
[24,103,105] and for reducing the phototoxicity of the free tetra-
pyrrolic intermediates of Chl formation. Seedlings grown in dark-
ness develop etioplasts from proplastids, which are transformed
into chloroplasts in light [24]. During etioplast development in dark-
ness, plastid number, plastid volume and expression of genes encod-
ing plastid polypeptides increases; however, expression of genes for
nuclear-encoded Chl a/b-binding antennae proteins and plastid-
A.N. Misra et al. / Nitric Oxide 39 (2014) 35–45 41
8. encoded Chl a-binding polypeptides is strictly light dependent
[107]. These photo-regulated processes have light receptors such
as phytochrome and cryptochrome [108,109]. NO stimulates the
accumulation of Chl during greening of leaves [19,71] and can imi-
tate red light responses in greening leaves [19]. The NO donor SNP
enhanced Chl synthesis and accumulation of LHCII and PSIA/B, the
effective quantum yield of PSII and maximal photochemical
efficiency of PSII of the developing chloroplasts during greening of
barley leaves [71]. The NO scavenger, 2-phenyl-4,4,5,5-tetramethy-
limidazoline-1-oxyl-3-oxide (PTIO), or NOS inhibitor, N-Omega-
nitro-L-arginine (L-NNA), retarded the greening process. Moreover,
sodium ferrocyanide, an analogue of SNP, nitrite and nitrate does
not have any effect on the greening process, suggesting a positive
role of NO in the greening process [71]; consistent with this sugges-
tion, endogenous NO content of greening leaves also increased in
parallel with the greening of leaves as measured by Chl accumula-
tion. [71]. Similarly, rice mutants (Osnoa1) with decreased NO
synthesis, possess less leaf chlorophyll than wild type plants [110]
(Table 3).
After the developmental and active photosynthetic phases of
the leaf comes the last phase, leaf senescence, when chlorophyll
is degraded through various enzymatic and oxidative processes
[105–109]. Both endogenous and exogenous NO at low concentra-
tions delay leaf senescence but at higher concentrations, which are
species specific, senescence is accelerated (for a review, see [111]).
Endogenous NO synthesis in leaves is known to regulate expres-
sion of the senescence associated gene 12 (sag12) in wild type as
well as transgenic plants through interaction with the plant hor-
mone, cytokinin, which was reported to act directly on chloroplasts
delaying the senescence process as measured by decrease in chlo-
rophyll [106]. However, when endogenous NO levels are lowered
by a genetic lesion in NO synthesis, leaf senescence is triggered
earlier than the wild type plants. NO can also prevent Fenton reac-
tion mediated generation of ROS, which accelerate leaf senescence
[111]. Exogenous polyamines rapidly induce NO production via yet
unknown reactions, and it has been proposed that both exogenous
NO and polyamines have senescence delaying effects in plants (for
a review, see [39]). NO deficiency in the Arabidopsis mutant (nos1/
noa1) was found to decrease the stability of photosynthetic com-
plexes in thylakoid membranes [112] thus demonstrating that
NO is a novel negative regulator of leaf senescence by delaying
chlorophyll catabolism and maintaining the stability of thylakoid
membranes.
Iron is a component of Fe–S cluster proteins of the photosyn-
thetic electron transport chain in the thylakoid membrane and is
involved in isocyclic ring formation, namely, the conversion of
Mg-protoporphrin monomethyl ester to protochlorophillide a in
the chlorophyll biosynthetic pathway, and consequently is essen-
tial for chloroplast development [24,112–116]. Recently, Kumar
et al. [117] suggested that NO increases the intracellular availabil-
ity of Fe by enhancing the chemical reduction of foliar Fe(III) to
Fe(II). Such NO-mediated intracellular Fe(II) availability to chloro-
plasts in green leaves under iron deficiency conditions could
consequently enable or accelerate the synthesis of chlorophyll
[118]. Electron micrographs of maize mesophyll cells from iron-
sparse maize plants reveal plastids with few thylakoid membranes
and rudimentary grana, while those from similar plants treated
with NO are as well developed as those from non-iron-stressed
plants [101]. This structural assembly of thylakoid membranes in
NO treated iron-sparse leaves is correlated with increased chloro-
phyll content which could induce the co-expression and stability
of both the D1 protein of PSII and the Rubisco L subunit [101].
Later, Graziano and Lamattina [119] proposed that NO may be
involved in mediating plant responses to iron deprivation, by act-
ing through the signal transduction pathway in root responses to
iron deficiency.
5.2. NO and abiotic stress
Several lines of evidence indicate that NO is involved in plant sig-
nal transduction in response to abiotic stress [8,120–125]. Under
abiotic stress conditions, NO eliminates the superoxide radicals,
reduces the toxicity of the ROS and as a signal molecule interacts
with plant hormones and ROS affecting physiological processes
[125–127]. NO may combine with O2
À
to form peroxynitrite
(ONOOÀ
), which has been reported to damage lipids, proteins and
nucleicacids [5]. There is evidence that NO influences plant response
to stressors such as drought, high and low temperature, salinity,
heavy metals, UV-B and oxidative stress [8,97]. The possible protec-
tive role of NO in oxidative stress was studied in Arabidopsis thaliana
mutant (Atnos1) with greatly diminished endogenous NO produc-
tion [128] (Table 3): the Atnos1 mutant showed increased hypersen-
sitivity to salt stress and methyl viologen treatment in comparison
with the wild type. It has been suggested that protection of plants
by NO under abiotic stress is most probably mediated by enhanced
activities of antioxidant enzymes [12]. Alternatively, the protective
action of NO on photosynthetic machinery against UV- and temper-
ature-stress may be mediated by influencing either the OEC complex
or the organization of PSII super-complex (LHCII–PSII complex). Our
studies, however, revealed different responses of the photosynthetic
apparatus to light, temperature stress and UV radiation stress which
were, in turn, dependent on the organization of the PSII complex
[129–135]. Other studies show a relationship between the organiza-
tion of the PSII complex and the oxidation state of the Mn clusters of
the OEC [134]: increasing of the PSII centres in S0 state, correlates
with the increase in the oligomerization of LHCII, and in turn
increases the stability of the PSII to abiotic stress [129–135]. Similar
changes in S0–S1 state distribution were observed after treatment of
the isolated thylakoid membranes with NO donor SNP [65], thus
suggesting a possible NO mediated mechanism for the protection
of photosynthetic machinery from abiotic stress. It has been shown
that treatment with the exogenous NO donor SNP induced abscisic
acid (ABA) biosynthesis during water stress [136,137], which could
be another example of protective action of NO against abiotic stress.
Also in biotic stress, NO has been reported to increase Chl content in
pea leaves and retard Chl degradation in Phytophthora-infected
Solanum tuborosum leaves [126]; however, studies on the
ameliorating effect of NO on biotic stress are otherwise limited to
hypersensitive reactions and scavenging of ROS.
We have summarized the data on exogenous NO-induced changes
of photosynthetic processes under physiological (non-stress)
conditions in Table 1 [23,29,56–58,62–65,70–72,76,81,82,84,85,
90–93,137–140] and in stress conditions in Table 2 [73,74,90,95,
99–101,138,141–150], respectively. Numerous recent reports are
cited and are concisely described in tabular form. These data show
Fig. 3. Schematic presentation of the possible target sites of NO action in the
chloroplast.
42 A.N. Misra et al. / Nitric Oxide 39 (2014) 35–45
9. the concentration-dependent effects of NO. The influence of NO on
plants using mutants with decreased endogenous NO production
also provided information on the effect of endogenous NO on
photosynthesis-parameters and photosynthetic complexes, as
described in the earlier sections, are also summarised in Table 3
[75,110,112,128].
6. Conclusion
This review shows that NO affects chloroplast structure and
function in vivo and in vitro irrespective of the plant source and
type of plant preparation used but the effects are different depen-
dent on concentration. The target sites of NO action on photosyn-
thetic apparatus in the chloroplast are summarized in Fig. 3. Data
revealed that at a relatively lower (nanomolar concentrations)
exogenous application and endogenously produced NO enhances
the photochemical efficiency, increases the net photosynthesis
and ameliorates the stress effects on chloroplasts. In stark contrast,
application of higher concentrations (micro to millimolar concen-
tration) of NO accelerates senescence or programmed cell death
in plants and may provide a natural pathway both to accelerate
the deterioration of chloroplasts and the degradation of plastid pig-
ments and so destroy photochemistry and photosynthesis. A closer
look at the available literature revealed that NO could act as a reg-
ulator of photosynthetic electron transport and that endogenous
NO generation in chloroplasts could regulate the activity of the
photosynthetic electron transport either by modifications of the
OEC and/or by activation of the cyclic electron transport around
PSII. On the other hand, under abiotic stress conditions, NO medi-
ated the protection of photosynthetic machinery.
Future studies need to discover more about the molecular basis
of NO action on physiological mechanisms in leaf cells and chloro-
plasts and the role of stressors and the endogenous signalling net-
work in triggering these NO effects.
Acknowledgments
This article is the result of cooperation under Bulgaria–India
Inter-Governmental Programme of Cooperation in Science and
Technology, project BIn-01/07 of the National Science Fund of Bul-
garia and INT/BULGARIA/B70/06 by Department of Science & Tech-
nology, Govt. of India. The financial support of UGC, India WoS
sanction No. F. 15-14/11 (SA-II) to MM and UGC MRP No. F. 36-
302/2008 to ANM is gratefully acknowledged. The authors are
thankful to Dr. Bryan Vought, Cambridge, MA, for their help in Eng-
lish corrections. We are grateful to Prof. Robert Porra, Australia, for
his intense reading of the manuscript and bringing it to this form of
readable English. Special thanks are due to Prof. Wah Soon Chow,
ANU, who is instrumental in helping us in completing the manu-
script in this form. We thank the reviewers for their immense
patience, critical comments and constructive criticisms.
References
[1] D. Wendehenne, A. Pugin, D. Klessig, J. Durner, Nitric oxide: comparative
synthesis and signaling in animal and plant cells, Trends Plant Sci. 6 (2001)
177–183.
[2] L. Lamattina, M.V. Beligni, C. Garcia-Mata, A.M. Laxalt, Method of enhancing
the metabolic function and the growing conditions of plants and seeds, US
Patent 6242384 (2001). <http://www.freepatentsonline.com/6242384.html>.
[3] K.J. Gupta, A.U. Igamberdiev, G. Manjunatha, S. Segu, J.F. Moran, B.
Neelawarne, H. Bauwe, W.M. Kaiser, The emerging roles of nitric oxide (NO)
in plant mitochondria, Plant Sci. 181 (2011) 520–526.
[4] D.A. Wink, J.B. Mitchell, Chemical biology of nitric oxide: insights into
regulatory, cytotoxic and cytoprotective mechanisms of nitric oxide, Free
Radic. Biol. Med. 25 (1998) 434–456.
[5] H. Yamasaki, Y. Sakihama, S. Takahashi, An alterative pathway for nitric oxide
production in plants: new features of an old enzyme, Trends Plant Sci. 4
(1999) 128–139.
[6] A.N. Misra, M. Misra, R. Singh, Nitric oxide biochemistry, mode of action and
signalling in plants, J. Med. Plants Res. 4 (2010) 2729–2749.
[7] A.N. Misra, M. Misra, R. Singh, Nitric oxide: a ubiquitous signaling molecule
with diverse role in plants, Afr. J. Plant Sci. 5 (2010) 57–74.
[8] A.N. Misra, M. Misra, R. Singh, Nitric oxide ameliorates stress responses in
plants, Plant Soil Environ. 57 (2011) 95–100.
[9] M. Misra, A.N. Misra, R. Singh, Nitric oxide signaling during senescence and
programmed cell death in leaves, in: D. Ekinci (Ed.), Chemical Biology,
Published by InTech, Rijeka, Croatia, 2012, pp. 159–186 (Chapter 9).
[10] S.J. Neill, R. Desikan, J.T. Hancock, Nitric oxide signalling in plants, New
Phytol. 159 (2003) 11–35.
[11] M. Delledonne, NO news is good news for plants, Curr. Opin. Plant Biol. 8
(2005) 390–396.
[12] S. Neill, R. Barros, J. Bright, R. Desikan, J. Hancock, J. Harrison, P. Morris, D.
Ribeiro, I. Wilson, Nitric oxide, stomatal closure and abiotic stress, J. Exp. Bot.
59 (2008) 165–176.
[13] N. Palavan-Unsal, D. Arisan, Nitric oxide signaling in plants, Bot. Res. 75
(2009) 203–229.
[14] Y.A. Krasylenko, A.I. Yemets, Y.B. Blume, Functional role of nitric oxide in
plants, Russ. J. Plant Physiol. 57 (2010) 451–461.
[15] M.V. Beligni, L. Lamattina, Is nitric oxide toxic or protective?, Trends Plant Sci
4 (1999) 299–300.
[16] M.V. Beligni, L. Lamattina, Nitric oxide in plants: the history is just beginning,
Plant Cell Environ. 24 (2001) 267–278.
[17] J.S. Stamler, D.J. Singel, J. Loscalzo, Biochemistry of nitric oxide and its redox-
activated forms, Science 258 (1992) 1898–1902.
[18] Y.Y. Leshem, R.B.H. Wills, V.V.-V. Ku, Evidence for the function of the free
radical gas – nitric oxide (NOÅ
) – as an endogenous maturation and
senescence regulating factor in higher plants, Plant Physiol. Biochem. 36
(1998) 825–833.
[19] M.V. Beligni, L. Lamattina, Nitric oxide stimulates seed germination and de-
etiolation, and inhibits hypocotyls elongation, three light-inducible responses
in plants, Planta 210 (2000) 215–221.
[20] M.V. Beligni, L. Lamattina, Nitric oxide: a non-traditional regulator of plant
growth, Trends Plant Sci. 6 (2001) 508–509.
[21] C. García-Mata, L. Lamattina, Nitric oxide induces stomatal closure and
enhances the adaptive plant responses against drought stress, Plant Physiol.
126 (2001) 1196–1204.
[22] Y.Y. Leshem, E. Haramaty, D. Iluz, Z. Malik, Y. Sofer, L. Roitman, Y. Leshem,
Effect of stress nitric oxide (NO): interaction between chlorophyll
fluorescence, galactolipid fluidity and lipoxygenase activity, Plant Physiol.
Biochem. 35 (1997) 573–579.
[23] A.C. Hill, J.H. Bennett, Inhibition of apparent photosynthesis by nitrogen
oxides, Atmos. Environ. 4 (1970) 341–348.
[24] P. Joshi, A.N. Misra, L. Nayak, B. Biswal, Response of mature, developing and
senescing chloroplast to environmental stress, in: B. Biswal, K. Krupinska, U.C.
Biswal (Eds.), Plastid Development in Leaves during Growth and Senescence,
in: Govindjee, T.D. Sharkey (Series Eds.), Advances in Photosynthesis and
Respiration, vol. 36, Springer, Dordrecht, 2013, pp. 641–668.
[25] A.N. Misra, A. Srivastava, R.J. Strasser, Utilization of fast chlorophyll a
fluorescence technique in assessing the salt/ion sensitivity of mung bean and
Brassica seedlings, J. Plant Physiol. 158 (2001) 1173–1181.
[26] A.N. Misra, A.K. Biswal, M. Misra, Physiological, biochemical and molecular
aspects of water stress responses in plants and the biotechnological
applications, Proc. Natl. Acad. Sci. (India) 72B (2002) 115–134.
[27] A. Galatro, S. Puntarulo, J.J. Guiamet, M. Simontacchi, Chloroplast
functionality has a positive effect on nitric oxide level in soybean
cotyledons, Plant Physiol. Biochem. 66 (2013) 26–33.
[28] R.K. Tewari, J. Prommer, M. Watanabe, Endogenous nitric oxide generation in
protoplast chloroplasts, Plant Cell Rep. 32 (2013) 31–44.
[29] S. Jasid, M. Simontacchi, C.G. Bartoli, S. Puntarulo, Chloroplasts as a nitric
oxide cellular source. Effect of reactive nitrogen species on chloroplastic
lipids and proteins, Plant Physiol. 142 (2006) 1246–1255.
[30] S. Hayat, S.A. Hasan, M. Mori, Q. Fariduddin, A. Ahmad, Nitric oxide:
chemistry, biosynthesis, and physiological role, in: S. Hayat, M. Mori, J.
Pichtel, A. Ahmad (Eds.), Nitric Oxide in Plant Physiology, Wiley-VCH Verlag
GmbH & Co., KGaA, Weinheim, 2010, pp. 1–15.
[31] M. Hasanuzzaman, M.A. Hossain, M. Fujita, Physiological and biochemical
mechanisms of nitric oxide induced abiotic stress tolerance in plants, Am. J.
Plant Physiol. 5 (2010) 295–324.
[32] L.A.J. Mur, J. Mandon, S.M. Cristescu, F.J.M. Harren, E. Prats, Methods of nitric
oxide detection in plants: a commentary, Plant Sci. 181 (2011) 509–519.
[33] L.A.J. Mur, J. Mandon, S. Persijn, S.M. Cristescu, I.E. Moshkov, G.V. Novikova,
M.A. Hall, F.J. Harren, K.H. Hebelstrup, K.J. Gupta, Nitric oxide in plants: an
assessment of the current state of knowledge, AoB Plants 5 (2013). pls052,
doi: 10.1093/aobpla/pls052.
[34] H.-T. Shi, R.-J. Li, W. Cai, W. Liu, Z.-W. Fu, Y.-T. Lu, In vivo role of nitric oxide in
plant response to abiotic and biotic stress, Plant Signal Behav. 7 (2012) 437–439.
[35] I. Saxena, G.S. Shekhawat, Nitric oxide (NO) in alleviation of heavy metal
induced phytotoxicity and its role in protein nitration, Nitric Oxide 32 (2013)
13–20.
[36] N. Nelson, C.F. Yocum, Structure and function of photosystems I and II, Annu.
Rev. Plant Biol. 57 (2006) 521–565.
[37] O. Nanba, K. Satoh, Isolation of a photosystem II reaction center consisting of
D-1 and D-2 polypeptides and cytochrome b-559, Proc. Natl. Acad. Sci. USA 84
(1987) 109–112.
A.N. Misra et al. / Nitric Oxide 39 (2014) 35–45 43
10. [38] A.K. Mattoo, J.B. Marder, M. Edelman, Dynamics of the photosystem II
reaction center, Cell 56 (1989) 241–246.
[39] A. Fröhlich, J. Durner, The hunt for plant nitric oxide synthase (NOS): is one
really needed?, Plant Sci 181 (2011) 401–404.
[40] K.J. Gupta, A. Zabalza, J.T. van Dongen, Regulation of respiration when the
oxygen availability changes, Physiol. Plant. 137 (2009) 383–391.
[41] F.J. Corpas, J.M. Palma, L.A. del Rio, J.B. Barroso, Evidence supporting the
existence of L-arginine-dependent nitric oxide synthase activity in plants,
New Phytol. 184 (2009) 9–14.
[42] W. Qiao, L.-M. Fan, Nitric oxide signaling in plant responses to abiotic
stresses, J. Integr. Plant Biol. 50 (2008) 1238–1246.
[43] H. Yamasaki, Nitrite dependent nitric oxide production pathway:
implications for involvement of active nitrogen species in photoinhibition
in vivo, Philos. Trans. R. Soc. Lond. 355 (2000) 1477–1488.
[44] R. Desikan, R. Griffiths, J. Hancock, S. Neill, New role for an old enzyme:
nitrate reductase-mediated nitric oxide generation is required for abscisic
acid-induced stomatal closure in Arabidopsis thaliana, Proc. Natl. Acad. Sci.
USA 99 (2002) 16314–16318.
[45] A. Sakamoto, M. Ueda, H. Morikawa, Arabidopsis glutathione-dependent
formaldehyde dehydrogenase is an S-nitrosoglutathione reductase, FEBS Lett.
515 (2002) 20–24.
[46] I. Foissner, D. Wendehenne, C. Langebartels, J. Durner, In vivo imaging of an
elicitor-induced nitric oxide burst in tobacco, Plant J. 23 (2000) 817–824.
[47] K.S. Gould, O. Lamotte, A. Klinger, A. Pugin, D. Wendehenne, Nitric oxide
production in tobacco leaf cells: a generalized stress response?, Plant, Cell
Environ 26 (2003) 1851–1862.
[48] N. Arnaud, I. Murgia, J. Boucherez, J.F. Briat, F. Cellier, F. Gaymard, An iron-
induced nitric oxide burst precedes ubiquitin-dependent protein degradation
for Arabidopsis AtFer1 ferritin gene expression, J. Biol. Chem. 281 (2006)
23579–23588.
[49] H. Ninnemann, J. Maier, Identifications for the occurrence of nitric oxide
synthases in fungi and plants and the involvement in photoconidiation of
Neurospora crassa, Photochem. Photobiol. 64 (1996) 393–398.
[50] N. Foresi, N. Correa-Aragunde, G. Parisi, G. Caló, G. Salerno, L. Lamattina,
Characterization of a nitric oxide synthase from the plant kingdom: NO
generation from the green alga Ostreococcus tauri is light irradiance and
growth phase dependent, Plant Cell 22 (2010) 3816–3830.
[51] F.Q. Guo, M. Okamoto, N.M. Crawford, Identification of a plant nitric oxide
synthase gene involved in hormonal signaling, Science 302 (2003) 100–103.
[52] N.M. Crawford, M. Gally, R. Tischner, Y.M. Heimer, M. Okamoto, A. Mack, Plant
nitric oxide synthase: back to square one, Trends Plant Sci. 11 (2006) 526–
527.
[53] T. Zemojtel, A. Fröhlich, M.C. Palmieri, M. Kolanczyk, I. Mikula, L.S. Wyrwicz,
E.E. Wanker, S. Mundlos, M. Vingron, P. Martasek, J. Durner, Plant nitric oxide
synthase: a never-ending story?, Trends Plant Sci 11 (2006) 524–525.
[54] P.C. Bethke, M.R. Badger, R.L. Jones, Apoplastic synthesis of nitric oxide by
plant tissues, Plant Cell 16 (2004) 332–341.
[55] R.V. Cooney, P.J. Harwood, L.J. Custer, A.A. Franke, Light-mediated conversion
of nitrogen dioxide to nitric oxide by carotenoids, Environ. Health Perspect.
102 (1994) 460–462.
[56] V. Petrouleas, B.A. Diner, Formation by NO of nitrosyl adducts of redox
components of the photosystem II reaction center. I. NO binds to the
acceptor-side non-heme iron, Biochim. Biophys. Acta 1015 (1990) 131–140.
[57] B.A. Diner, V. Petrouleas, Formation by NO of nitrosyl adducts of redox
components of the photosystem II reaction center. II: evidence that HCO3
À
/
CO2 binds to the acceptor-side non-heme iron, Biochim. Biophys. Acta 1015
(1990) 141–149.
[58] G. Schansker, C. Goussias, V. Petrouleas, A.W. Rutherford, Reduction of the Mn
cluster of the water-oxidizing enzyme by nitric oxide: formation of an SÀ2
state, Biochemistry 41 (2002) 3057–3064.
[59] Y. Sanakis, C. Goussias, R.P. Mason, V. Petrouleas, NO interacts with the
tyrosine radical YD
Å
of photosystem II to form an iminoxyl radical,
Biochemistry 36 (1997) 1411–1417.
[60] C. Goussias, Y. Deligiannakis, Y. Sanakis, N. Ioannidis, V. Petrouleas, Probing
subtle coordination changes in the iron–quinone complex of photosystem II
during charges separation by the use of NO, Biochemistry 41 (2002) 15212–
15223.
[61] J.J.S. Van Rensen, J.F.H. Snel, Regulation of photosynthetic electron transport
by bicarbonate, formate and herbicides in isolated broken and intact
chloroplasts, Photosynth. Res. 6 (1985) 231–246.
[62] B. Wodala, Z. Deák, I. Vass, L. Erdei, I. Altorjay, F. Horváth, In vivo target sites
of NO in photosynthetic electron transport as studied by chlorophyll
fluorescence in pea leaves, Plant Physiol. 146 (2008) 1920–1927.
[63] B. Wodala, A. Ördög, F. Horváth, The cost and risk of using sodium
nitroprusside as a NO donor in chlorophyll fluorescence experiments, J.
Plant Physiol. 167 (2010) 1109–1111.
[64] B. Wodala, Z. Deák, I. Vass, L. Erdei, F. Horváth, Nitric oxide modifies
photosynthetic electron transport in pea leaves, Acta Biol. Szeg. 49 (2005) 7–
8.
[65] R. Vladkova, A.G. Dobrikova, R. Singh, A.N. Misra, E. Apostolova, Photoelectron
transport ability of chloroplast thylakoid membranes treated with NO donor
SNP: changes in flash oxygen evolution and chlorophyll fluorescence, Nitric
Oxide 24 (2011) 84–90.
[66] E.H. Murchie, T. Lawson, Chlorophyll fluorescence analysis: a guide to good
practice and understanding some new applications, J. Exp. Bot. 64 (2013)
3983–3998.
[67] A.N. Misra, M. Misra, R. Singh, Chlorophyll fluorescence in plant biology, in:
A.N. Misra (Ed.), Biophysics, InTech Publ., 2012, pp. 171–192 (Chapter 7).
[68] N.R. Baker, Chlorophyll fluorescence: a probe of photosynthesis in vivo, Annu.
Rev. Plant Biol. 59 (2008) 89–113.
[69] M.M. Najafpour, A.N. Moghaddam, Y.N. Yang, E.M. Aro, R. Carpentier, J.J.
Eaton-Rye, C.-H. Lee, S.I. Allakhverdiev, Biological water-oxidizing complex: a
nano-sized manganese–calcium oxide in a protein environment, Photosynth.
Res. 114 (2012) 1–13.
[70] J.D. Yang, H.L. Zhao, T.H. Zhang, J.F. Yun, Effects of exogenous nitric oxide on
photochemical activity of photosystem II in potato leaf tissue under non-
stress condition, Acta Bot. Sin. 46 (2004) 1009–1014.
[71] L. Zhang, Y. Wang, L. Zhao, S. Shi, L. Zhang, Involvement of nitric oxide in
light-mediated greening of barley seedlings, J. Plant Physiol. 163 (2006) 818–
826.
[72] S. Takahashi, H. Yamasaki, Reversible inhibition of photophosphorylation in
chloroplasts by nitric oxide, FEBS Lett. 512 (2002) 145–148.
[73] S. Shi, G. Wang, Y. Wang, L. Zhang, L. Zhang, Protective effect of nitric oxide
against oxidative stress under ultraviolet-B radiation, Nitric Oxide 13 (2005)
1–9.
[74] X.W. Zhang, Y.J. Dong, X.K. Qiu, G.Q. Hu, Y.H. Wang, Q.H. Wang, Exogenous
nitric oxide alleviates iron-deficiency chlorosis in peanut growing on
calcareous soil, Plant Soil Environ. 58 (2012) 111–120.
[75] H. Liu, E. Lau, M.P.Y. Lam, H. Chu, S. Li, G. Huang, P. Guo, J. Wang, L. Jiang, I.K.
Chu, C. Lo, Y. Tao, OsNOA1/RIF1 is a functional homolog of AtNOA1/RIF1:
implication for a highly conserved plant cGTPase essential for chloroplast
function, New Phytol. 187 (2010) 83–105.
[76] A. Ördög, B. Wodala, T. Rózsavölgyi, I. Tari, F. Horváth, Regulation of guard cell
photosynthetic electron transport by nitric oxide, J. Exp. Bot. 64 (2013) 1357–
1366.
[77] C.W. Hoganson, G.T. Babcock, A metaloradical mechanism for the generation
of oxygen from water in photosynthesis, Science 277 (1997) 1953–1956.
[78] M. Haumann, P. Liebisch, C. Müller, M. Barra, M. Grabolle, H. Dau,
Photosynthetic O2 formation tracked by time-resolved X-ray experiments,
Science 310 (2005) 1019–1021.
[79] C. Goussias, N. Ioannidis, V. Petrouleas, Low-temperature interactions of NO
with the S1 and S2 states of the water-oxidizing complex of photosystem II. A
novel Mn-multiline EPR signal derived from the S1 state, Biochemistry 36
(1997) 9261–9266.
[80] J. Sarrou, N. Ioannidis, Y. Deligiannakis, V. Petrouleas, A Mn(II)ÀMn(III)
EPR signal arises from the interaction of NO with the S1 state of the
water-oxidizing complex of photosystem II, Biochemistry 37 (1998)
3581–3587.
[81] J. Sarrou, S. Isgandarova, J. Kern, A. Zouni, G. Renger, W. Lubitz, J. Messinger,
Nitric oxide-induced formation of the SÀ2 state in the oxygen-evolving
complex of photosystem II from Synechococcus elongatus, Biochemistry 42
(2003) 1016–1023.
[82] N. Ioannidis, G. Schansker, V.V. Barynin, V. Petrouleas, Interaction of nitric
oxide with the oxygen evolving complex of photosystem II and manganese
catalase: a comparative study, J. Biol. Inorg. Chem. 5 (2000) 354–363.
[83] S. Styring, A.W. Rutherford, In the oxygen-evolving complex of photosystem II
the S0 state is oxidized to the S1 state by D+
(signal II slow), Biochemistry 26
(1987) 2401–2405.
[84] H.K. Lum, C.H. Lee, Y.K. Butt, S.C. Lo, Sodium nitroprusside affects the level of
photosynthetic enzymes and glucose metabolism in Phaseolus aureus (mung
bean), Nitric Oxide 12 (2005) 220–230.
[85] B. Wodala, F. Horváth, The effect of exogenous NO on PSI photochemistry in
intact pea leaves, Acta Biol. Szeg. 52 (2008) 243–245.
[86] D. Stroebel, Y. Choquet, J.L. Popot, D. Picot, An atypical haem in the
cytochrome b6f complex, Nature 426 (2003) 413–418.
[87] G. Kurisu, H.M. Zhang, J.L. Smith, W.A. Cramer, Structure of the cytochrome
b6f complex of oxygenic photosynthesis: tuning the cavity, Science 302
(2003) 1009–1014.
[88] A.I. Twigg, D. Baniulis, W.A. Cramer, M.P. Hendrich, EPR detection of an O2
surrogate bound to heme cn of the cytochrome b6f complex, J. Am. Chem. Soc.
131 (2009) 12536–12537.
[89] P.S. Brookes, D.W. Kraus, S. Shiva, J.E. Doeller, M.C. Barone, R.P. Patel, J.R.
Lancaster Jr., V. Darley-Usmar, Control of mitochondrial respiration by NOÅ
,
effects of low oxygen and respiratory state, J. Biol. Chem. 278 (2003) 31603–
31609.
[90] L. Yang, R. Han, Y. Sun, Effects of exogenous nitric oxide on wheat exposed to
enhanced ultraviolet-B radiation, Am. J. Plant Sci. 4 (2013) 1285–1290.
[91] J. Tu, W.B. Shen, L.L. Xu, Regulation of nitric oxide on the aging process of
wheat leaves, Acta Bot. Sin. 45 (2003) 1055–1062.
[92] J.K. Abat, A.K. Mattoo, R. Deswal, S-nitrosylated proteins of a medicinal CAM
plant Kalanchoe pinnata-ribulose-1,5-bisphosphate carboxylase/oxygenase
activity targeted for inhibition, FEBS J. 275 (2008) 2862–2872.
[93] C. Lindermayr, G. Saalbach, J. Durner, Proteomic identification of S-
nitrosylated proteins in Arabidopsis, Plant Physiol. 137 (2005) 921–930.
[94] B.-W. Yun, A. Feechan, M. Yin, N.B.B. Saidi, T. Le Bihan, M. Yu, J.W. Moore, J.-G.
Kang, E. Kwon, S.H. Spoel, J.A. Pallas, G.J. Loake, et al., S-nitrosylation of
NADPH oxidase regulates cell death in plant immunity, Nature 478 (2011)
264–268.
[95] G. Tanou, C. Job, L. Rajjou, E. Arc, M. Belghazi, G. Diamantidis, A. Molassiotis,
D. Job, Proteomics reveals the overlapping roles of hydrogen peroxide and
nitric oxide in the acclimation of Citrus plants to salinity, Plant J. 60 (2009)
795–804.
44 A.N. Misra et al. / Nitric Oxide 39 (2014) 35–45
11. [96] A. Polverari, B. Molesini, M. Pezzotti, R. Buonaurio, M. Marte, M. Delledonne,
Nitric oxide-mediated transcriptional changes in Arabidopsis thaliana, Mol.
Plant Microbe Interact. 16 (2003) 1094–1105.
[97] M. Arasimowicz, J. Floryszak-Wieczorek, Nitric oxide as a bioactive signalling
molecule in plant stress responses, Plant Sci. 172 (2007) 876–887.
[98] S. Grun, C. Lindermayr, S. Sell, J. Durner, Nitric oxide and gene regulation in
plants, J. Exp. Bot. 57 (2006) 507–516.
[99] M.V. Beligni, L. Lamattina, Nitric oxide interferes with plant photooxidative
stress by detoxifying reactive oxygen species, Plant, Cell Environ. 25 (2002)
737–748.
[100] H. Qian, W. Chen, J. Li, J. Wang, Z. Zhou, W. Liu, W. Fu, The effect of exogenous
nitric oxide on alleviating herbicide damage in Chlorella vulgaris, Aquat.
Toxicol. 92 (2009) 250–257.
[101] M. Graziano, M.V. Beligni, L. Lamattina, Nitric oxide improves internal iron
availability in plants, Plant Physiol. 130 (2002) 1852–1859.
[102] J. Wildt, D. Kley, A. Rockel, P. Rockel, H.J. Segschneider, Emission of NO from
several higher plant species, J. Geophys. Res. 102 (1997) 5919–5927.
[103] F. Dilnawaz, P. Mahapatra, M. Misra, N.K. Ramaswamy, A.N. Misra, The
distinctive pattern of phtosystem 2 activity, photosynthetic pigment
accumulation and ribulose-1,5-bisphosphate carboxylase/oxygenase
content of chloroplasts along the axis of primary wheat leaf lamina,
Photosynthetica 39 (2001) 557–563.
[104] A.K. Biswal, F. Dilnawaz, K.A.V. David, N.K. Ramaswamy, A.N. Misra, Increase
in the intensity of thermoluminescence Q-band during leaf ageing is due to a
block in the electron transfer from QA to QB, Luminescence 16 (2001) 309–
313.
[105] A.N. Misra, M. Misra, Effect of age and rehydration on greening of wheat
leaves, Plant Cell Physiol. 28 (1987) 47–51.
[106] A.N. Misra, U.C. Biswal, Effect of phytohormones on the chlorophyll degradation
during aging of chloroplasts in vivo and in vitro, Protoplasma 105 (1980) 1–8.
[107] R.R. Klein, J.E. Mullet, Regulation of chloroplast-encoded chlorophyll-binding
protein translation during higher plant chloroplast biogenesis, J. Biol. Chem.
26 (1986) 11138–11145.
[108] B.J. Pogson, V. Albrecht, Genetic dissection of chloroplast biogenesis and
development: an overview, Plant Physiol. 155 (2011) 1545–1551.
[109] P. Matile, S. Hortensteiner, H. Thomas, B. Krautler, Chlorophyll breakdown in
senescent leaves, Plant Physiol. 112 (1996) 1403–1409.
[110] Q. Yang, H. He, H. Li, H. Tian, J. Zhang, L. Zhai, J. Chen, H. Wu, G. Yi, Z.-H. He, X.
Peng, NOA1 functions in a temperature-dependent manner to regulate
chlorophyll biosynthesis and Rubisco formation in rice, PLoS ONE 6 (2011)
e20015, http://dx.doi.org/10.1371/journal.pone.0020015.
[111] D. Procházková, N. Wilhelmová, Nitric oxide, reactive nitrogen species and
associated enzymes during plant senescence, Nitric Oxide 24 (2011) 61–65.
[112] F. Liu, F.-Q. Guo, Nitric oxide deficiency accelerates chlorophyll breakdown
and stability loss of thylakoid membranes during dark-induced leaf
senescence in Arabidopsis, PLoS ONE 8 (2013) e56345, http://dx.doi.org/
10.1371/journal.pone.0056345.
[113] P. Brzezowski, B. Grimm, Chlorophyll Metabolism, eLS (2013). doi: 10.1002/
9780470015902.a0020084.pub2.
[114] A. Larbi, A. Abadia, J. Abadia, F. Morales, Down co-regulation of light
absorption, photochemistry, and carboxylation in Fe-deficient plants growing
in different environments, Photosynth. Res. 89 (2006) 113–126.
[115] H. Lambers, F.S. Chapin III, T.L. Pons, Plant Physiological Ecology, second ed.,
Springer, New York, 2008.
[116] H. Ye, M. Pilon, E.A.H. Pilon-Smits, CpNifS-dependent iron-sulfur cluster
biogenesis in chloroplasts, New Phytol. 171 (2006) 285–292.
[117] P. Kumar, R.K. Tewari, P.N. Sharma, Sodium nitroprusside-mediated
alleviation of iron deficiency and modulation of antioxidant responses in
maize plants, AoB Plants (2010) plq002. doi: 10.1093/aobpla/plq002.
[118] Z.E.L. Dina, A.K. Albidin, Role of nitric oxide on iron homeostasis, chlorophyll
biosynthesis and antioxidants system in two wheat cultivars, Am. J. Plant
Physiol. 2 (2007) 237–250.
[119] M. Graziano, L. Lamattina, Nitric oxide accumulation is required for
molecular and physiological responses to iron deficiency in tomato roots,
Plant J. 52 (2007) 949–960.
[120] X. Tian, Y. Lei, Nitric oxide treatment alleviates drought stress in wheat
seedlings, Biol. Plant. 50 (2006) 775–778.
[121] J. Sun, H. Jiang, Y. Xu, H. Li, X. Wu, Q. Xie, C. Li, The CCCH-type zinc finger
proteins AtSZF1 and AtSZF2 regulate salt stress responses in Arabidopsis,
Plant Cell Physiol. 48 (2007) 1148–1158.
[122] F. Zhang, Y. Wang, Y. Yang, H. Wu, W. Di, J. Liu, Involvement of hydrogen
peroxide and nitric oxide in salt resistance in the calluses from Populus
euphratica, Plant, Cell Environ. 30 (2007) 775–785.
[123] M. Arasimowicz-Jelonek, J. Floryszak-Wieczorek, J. Deckert, R. Rucin´ ska-
Sobkowiak, J. Gzyla, S. Pawlak-Sprada, D. Abramowski, T. Jelonek, E.A.
Gwózdz, Nitric oxide implication in cadmium-induced programmed cell
death in roots and signaling response of yellow lupine plants, Plant Physiol.
Biochem. 58 (2012) 124–134.
[124] M. Farooq, S.M.A. Basra, A. Wahid, H. Rehman, Exogenously applied nitric
oxide enhances the drought tolerance in fine grain aromatic rice (Oryza sativa
L.), J. Agron. Crop Sci. 195 (2009) 254–261.
[125] A. Sehrawat, R. Gupta, R. Deswal, Nitric oxide-cold stress signalling cross-talk,
evolution of a novel regulatory mechanism, Proteomics 13 (2013) 1816–
1835, http://dx.doi.org/10.1002/pmic.201200445.
[126] A.M. Laxalt, M.V. Beligni, L. Lamattina, Nitric oxide preserves the level of
chlorophyll in potato leaves infected by Phytophthora infestans, Eur. J. Plant
Pathol. 103 (1997) 643–651.
[127] L. Zhao, F. Zhang, J. Guo, Y. Yang, B. Li, L. Zhang, Nitric oxide functions as a
signal in salt resistance in the calluses from two ecotypes of reed, Plant
Physiol. 134 (2004) 849–857.
[128] M. Zhao, X. Zhao, Y. Wu, L. Zhang, Enhanced sensitivity to oxidative stress in
an Arabidopsis nitric oxide synthase mutant, J. Plant Physiol. 164 (2007) 737–
745.
[129] P.I. Ivanova, A.G. Dobrikova, S.G. Taneva, E.L. Apostolova, Sensitivity of the
photosynthetic apparatus to UV-A radiation: a role of light-harvesting
complex II – photosystem II supercomplex organization, Radiat. Environ.
Biophys. 47 (2008) 169–177.
[130] K. Dankov, A. Dobrikova, B. Bogos, Z. Gombos, E. Apostolova, The role of
anionic lipids in LHCII organization and in photoinhibition of photosynthetic
apparatus, Comp. Rend. Acad. Bulg. Sci. 62 (2009) 941–948.
[131] K. Dankov, S.G. Taneva, E.L. Apostolova, Freeze–thaw damage of
photosynthetic apparatus. Effect of the organization of LHCII–PSII
supercomplex, Comp. Rend. Acad. Bulg. Sci. 62 (2009) 1103–1110.
[132] K.G. Dankov, A.G. Dobrikova, B. Ughy, B. Bogos, Z. Gombos, E.L. Apostolova,
LHCII organization and thylakoid lipids affect the sensitivity of the
photosynthetic apparatus to high-light treatment, Plant Physiol. Biochem.
49 (2011) 629–635.
[133] E.L. Apostolova, A.G. Dobrikova, Effect of high temperature and UV-A
radiation on the photosystem II, in: M. Pessarakli (Ed.), Handbook of Plant
and Crop Stress, third ed., Taylor & Francis Group, 2010, pp. 577–591.
[134] E.L. Apostolova, A.G. Dobrikova, P.I. Ivanova, I.B. Petkanchin, S.G. Taneva,
Relationship between the organization of the PSII supercomplex and the
functions of the photosynthetic apparatus, J. Photochem. Photobiol., B 83
(2006) 114–122.
[135] E.L. Apostolova, A.N. Misra, Alterations in structural organization affect the
functional ability of photosynthetic apparatus, in: M. Pessarakli (Ed.),
Handbook of Plant and Crop Physiology, expanded third ed., CRC Press,
Taylor & Francis Group, 2014, pp. 103–120 (Chapter 3).
[136] Z. Zhao, G. Chen, C. Zhang, Interaction between reactive oxygen species and
nitric oxide in drought-induced abscisic acid synthesis in root tips of wheat
seedlings, Aust. J. Plant Physiol. 28 (2001) 1055–1061.
[137] H. Xing, L. Tan, L. An, Z. Zhao, S. Wang, C. Zhang, Evidence for the involvement
of nitric oxide and reactive oxygen species in osmotic stress tolerance of
wheat seedlings: inverse correlation between leaf abscisic acid accumulation
and leaf water loss, Plant Growth Reg. 42 (2004) 61–68.
[138] X.-X. Wu, H.-D. Ding, J.-L. Chen, H.-J. Zhang, W.-M. Zhu, Attenuation of salt-
induced changes in photosynthesis by exogenous nitric oxide in tomato
(Lycopersicon esculentum Mill. L.) seedlings, Afr. J. Biotechnol. 9 (2010) 7837–7846.
[139] P. Filippou, C. Antoniou, V. Fotopoulos, The nitric oxide donor sodium
nitroprusside regulates polyamine and proline metabolism in leaves of
Medicago truncatula plants, Free Radic. Biol. Med. 56 (2013) 172–183.
[140] I.S. Booij-James, M. Edelman, A.K. Mattoo, Nitric oxide donor-mediated
inhibition of phosphorylation shows that light-mediated degradation of
photosystem II D1 protein and phosphorylation are not tightly linked, Planta
229 (2009) 1347–1352.
[141] J.D. Yang, J.Y. Yun, T.H. Zhang, H.L. Zhao, Presoaking with nitric oxide donor
SNP alleviates heat shock damages in mung bean leaf discs, Bot. Stud. 47
(2006) 129–136.
[142] W. Yang, Y. Sun, S. Chen, J. Jiang, F. Chen, W. Fang, Z. Liu, The effect of
exogenously applied nitric oxide on photosynthesis and antioxidant activity
in heat stressed chrysanthemum, Biol. Plant. 55 (2011) 737–740.
[143] H. Fan, S. Guo, Y. Jiao, R. Zhang, J. Li, Effects of exogenous nitric oxide on
growth, active oxygen species metabolism, and photosynthetic
characteristics in cucumber seedlings under NaCl stress, Front. Agric. China
1 (2007) 308–314.
[144] A. Uchida, A.T. Jagendorf, T. Hibino, T. Takabe, T. Takabe, Effects of hydrogen
peroxide and nitric oxide on both salt and heat stress tolerance in rice, Plant
Sci. 163 (2002) 515–523.
[145] L.-T. Yang, Y.-P. Qi, L.-S. Chen, W. Sang, X.-J. Lin, Y.-L. Wu, C.-J. Yang, Nitric oxide
protects sour pummelo (Citrus grandis) seedlings against aluminum-induced
inhibition of growth and photosynthesis, Environ. Exp. Bot. 82 (2012) 1–13.
[146] F. Chen, F. Wang, H. Sun, Y. Cai, W. Mao, G. Zhang, E. Vincze, F. Wu, Genotype-
dependent effect of exogenous nitric oxide on Cd-induced changes in
antioxidative metabolism, ultrastructure, and photosynthetic performance in
barley seedlings (Hordeum vulgare), J. Plant Growth Regul. 29 (2010) 394–408.
[147] J. Xiong, L. An, H. Lu, C. Zhu, Exogenous nitric oxide enhances cadmium
tolerance of rice by increasing pectin and hemicelluloses contents in root cell
wall, Planta 230 (2009) 755–765.
[148] T.-Y. Kim, M.-H. Jo, J.-H. Hong, Protective effect of nitric oxide against
oxidative stress under UV-B radiation in maize leaves, J. Environ. Sci. 19
(2010) 1323–1334.
[149] R.-X. Shao, Q.-H. Yang, L.-F. Xin, Z.-P. Shangguan, Effects of exogenous nitric
oxide and cytokinin on the growth and photosynthesis of wheat seedlings
under water deficit, J. Food Agric. Environ. 10 (2012) 1451–1456.
[150] J. Tan, H. Zhao, J. Hong, Y. Han, H. Li, W. Zhao, Effects of exogenous nitric
oxide on photosynthesis, antioxidant capacity and proline accumulation in
wheat seedlings subjected to osmotic stress, World J. Agric. Sci. 4 (2008) 307–
313.
A.N. Misra et al. / Nitric Oxide 39 (2014) 35–45 45
![Review
Action and target sites of nitric oxide in chloroplasts
Amarendra N. Misra a,⇑
, Radka Vladkova b
, Ranjeet Singh a
, Meena Misra a
,
Anelia G. Dobrikova b
, Emilia L. Apostolova b
a
Centre for Life Sciences, School of Natural Sciences, Central University of Jharkhand, Ratu Lohardaga Road, Brambe, Ranchi 435020, India
b
Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl.21, Sofia 1113, Bulgaria
a r t i c l e i n f o
Article history:
Received 28 June 2013
Revised 17 March 2014
Available online 13 April 2014
Keywords:
Nitric oxide
Chloroplast
Photosynthetic electron transport
Oxygen evolution
Photophosphorylation
a b s t r a c t
Nitric oxide (NO) is an important signalling molecule in plants under physiological and stress conditions.
Here we review the influence of NO on chloroplasts which can be directly induced by interaction with the
photosynthetic apparatus by influencing photophosphorylation, electron transport activity and oxido-
reduction state of the Mn clusters of the oxygen-evolving complex or by changes in gene expression.
The influence of NO-induced changes in the photosynthetic apparatus on its functions and sensitivity
to stress factors are discussed.
Ó 2014 Elsevier Inc. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2. Chloroplasts generate NO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3. Target sites of NO action in chloroplasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.1. Effects of NO on photosystem II. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.1.1. Electron transport chain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.1.2. Effects of NO on PSII photochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.1.3. Oxygen evolving complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.2. Effect of NO on the photosystem I activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.3. Interaction of NO with cytochrome b6f complex. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.4. Photophosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.5. Modulation of Carbon dioxide reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
4. Regulation of plastid gene expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
5. Effect of NO on photosynthesis under physiological and stress conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
5.1. Development and senescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
5.2. NO and abiotic stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
http://dx.doi.org/10.1016/j.niox.2014.04.003
1089-8603/Ó 2014 Elsevier Inc. All rights reserved.
Abbreviations: Chl, chlorophyll; cPTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide; cyt b6f, cytochrome b6f; ETR, electron transport rate through
PSII; Fm/Fo, ratio of the maximal to minimal fluorescence in dark adapted state; Fv/Fm, maximal quantum efficiency of PSII photochemistry in dark adapted state; Fv0
/Fm0
,
efficiency of excitation energy capture by open PSII reaction centres; UPSII, effective quantum yield (efficiency) of PSII photochemistry in light adapted state; GSNO, S-
nitrosoglutathione; GSSG, glutathione disulphide; NO, nitric oxide; NOR2, (±)-(E)-4-methyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide; NOS, nitric oxide synthase; NPQ,
non-photochemical quenching; NR, nitrate reductase; OEC, oxygen evolving complex; PAM, pulse amplitude modulation; Pn, net photosynthetic rate; PPFD,
photosynthetically active photon flux density; PSI, photosystem I; PSII, photosystem II; qL, coefficient of photochemical fluorescence quenching assuming interconnected
PSII antennae; qN, non-photochemical quenching coefficient; qP, coefficient of photochemical fluorescence quenching assuming non-interconnected PSII antennae; ROS,
reactive oxygen species; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; SNAP, S-nitroso-N-acetylpenicillinamine; SIN-1, 3-morpholinosydnonimine; SNOC, S-
nitrosocysteine; SNP, sodiumnitroprusside.
⇑ Corresponding author. Fax: +91 6531 294161.
E-mail address: misraan@yahoo.co.uk (A.N. Misra).
Nitric Oxide 39 (2014) 35–45
Contents lists available at ScienceDirect
Nitric Oxide
journal homepage: www.elsevier.com/locate/yniox](https://image.slidesharecdn.com/6222b484-7e41-4ac3-9d32-50299fb0b2ea-150923095341-lva1-app6891/85/misra-et-al-2014-1-320.jpg)
![1. Introduction
Nitric oxide (NO) is now established as an important signalling
molecule regulating a wide range of physiological, biochemical and
molecular processes in animals [1]. The pathway of NO synthesis in
animals, mediated by isoforms of the enzyme nitric oxide synthase
(NOS) from L-arginine, is well characterized. In animals, NO plays a
vital role in maintaining blood pressure, host immune system, neu-
ral transmission and gene expression [2]. Nitric oxide is a gaseous
free radical molecule which diffuses readily through biological
membranes and has a biological half-life ranging from 5 to 13 s
[3]. This short half-life reflects the highly reactive nature of the
molecule: it reacts with metal complexes and other radicals, and
with biomolecules such as nucleic acids, proteins and lipids [4].
It can also react with superoxide (O2
À
) to form peroxynitrite
(ONOOÀ
), which can damage lipids, proteins and nucleic acids
[5]. There is growing evidence that NO is also an important signal-
ling molecule in plants [2,6–11] which has been obtained via the
application of NO, usually in the form of donors, via the measure-
ment of endogenous NO, and through the manipulation of content
by chemical and genetic means [12]. As in animals, the roles of NO
in plants may be equally diverse by having a role in cell signalling
and regulation of plant growth and development [6–10,13]. These
effects can be manifested directly by exogenous NO donors or
through effector molecules that preferentially regulate the redox
state of the cell [14]. The latter process occurs by the endogenous
generation of NO in plants either by metabolic regulations, pertur-
bations or modulations by developmental, environmental or
genetic cues [6–9]. NO can modify the structure and function of
haemoproteins or proteins by reversible iron-ligand binding to
functional haem prosthetic groups or to thiol groups and its role
as a radical cytotoxin or cytoprotectant through the redox homeo-
stasis of the cellular environment in plants has also been reported
[15–17]. These diverse effects of NO arise from concentration-
dependent reactions in living cells and tissue: low (sub-lmolar
concentrations) of exogenous NO donors stimulate plant growth,
regulation of stomatal movement and retardation of programmed
cell death and senescence [6–9,16,18–21] while higher (mmolar
concentrations) disturb metabolic activities in plant cells including
decreased photosynthetic electron transport, increased viscosity of
isolated thylakoid membrane lipid monolayers, leaf expansion
[18,22] and net photosynthesis [23].
Chloroplasts are very sensitive and susceptible to stressful cli-
matic changes [24–26] and because NO is a signalling molecule
in plants known to ameliorate stress effects, understanding its role
on chloroplasts is crucial. In addition, chloroplasts are proposed as
a site for the synthesis of endogenous nitric oxide [27–29]. Cur-
rently, despite many recent reviews on NO effects in plants [6–
9,14,30–35], little is known of the effect of NO on the regulation
of different physiological, biochemical and molecular processes in
chloroplasts. This review summarizes the most recent studies on
the effect of NO on chloroplasts with particular emphasis on the
effect of NO on the efficiency of the photochemical-transduction-
and carbon-fixation – pathways as well as its stress ameliorating
effect on chloroplasts.
2. Chloroplasts generate NO
Chloroplasts are semi-autonomous organelles in green plants
with diverse structure, function and environmental adaptability.
They have developed a thylakoid membrane network, suspended
in the stroma matrix: these membranes possess pigment protein
complexes which transduce solar energy by converting it to electri-
cal potentials that generate chemical energy (ATP) and reducing
equivalents (protons) in the form of NADPH+
generated by the
photolysis of water. The ATP and reductants generated are utilized
to reduce carbon dioxide to carbohydrates in the stroma.
The thylakoid membranes have four multi-subunit protein
complexes: photosystem II (PSII), photosystem I (PSI), cytochrome
b6f (cyt b6f) and ATP-synthase [36]. PSII and PSI are intrinsic pig-
ment–protein complexes spatially separated in the stacked (grana)
and unstacked (stromal lamellae) regions of the thylakoid mem-
branes in chloroplasts. PSII catalyzes the oxidation of water and
the reduction of plastoquinone [37,38]. Cyt b6f complex accepts
an electron from plastoquinol and donates it to PSI via plastocya-
nin. The electron is transferred ultimately to the NADPH+
via PSI,
and then released to the stroma to be utilized in the CO2 reduction
process. Concomitantly with the electron transfer, the accumulated
protons in the lumen are used for ATP synthesis by ATPase.
Production of NO in plant cells arises from several different
pathways and in different organelles [30,39,40] including mito-
chondria [40], peroxisomes [41] and the chloroplast [27,28].
Dependant on concentration, NO can provoke both beneficial and
harmful effects which also depend on its location within the plant
cell [42,43]. In plants, NO can be produced from nitrite [44], from L-
arginine by NOS-like biochemical pathways [41] and from S-nitro-
soglutathione by decomposition [45]. Endogenous NO generation
in chloroplasts without any external donors has been recently
reported [27,28]. It is notable that NO synthesis was first detected
in chloroplasts in response to various stressors like high tempera-
ture, salinity, iron, elicitors or osmotic stress, using NO-sensitive
diaminofluorescein probes [46–48].
Nitric oxide synthase (NOS) activity was first detected in fungi
and a higher plant by Ninnemann and Maier [49]. Despite evidence
of an Arg-dependent pathway for NO synthesis in higher plants, no
NOS homologs have been identified in plants [50]. Guo et al. [51]
described the identification of a plant NOS gene involved in hor-
monal signalling (Atnos1); however, since no NOS activity was
detected in the purified AtNOS1 protein [52,53], it was renamed
AtNOA1 (nitric oxide associated 1), because it appeared essential
for NO generation in the cell. Another enzymatic source of NO in
plants is the cytosolic nitrate reductase (NR) [12] and the NO so
produced can readily diffuse into the chloroplast stroma [43].
Nitrite can also be reduced to NO non-enzymatically at acidic pH
values [54]. Non-enzymatic, light-mediated conversion of nitrogen
dioxide to nitric oxide by the plastid carotenoids has also been doc-
umented [55].
3. Target sites of NO action in chloroplasts
The effect of NO on target sites in the chloroplast and on its pho-
tosynthetic apparatus has mostly been accumulated by applying
exogenous gaseous NO or by various NO donors on leaves, leaf
discs, leaf extracts, intact chloroplasts or isolated chloroplast com-
ponents. The results are often confusing and contradictory but, in
this review, an effort is made to clarify such discrepancies.
3.1. Effects of NO on photosystem II
3.1.1. Electron transport chain
Several electron transport chain components of PSII have been
identified as target sites for NO in chloroplasts. EPR and chloro-
phyll fluorescence analysis of isolated PSII-enriched membranes
and chloroplasts treated with NO gas demonstrated NO binding
to the non-haem iron of PSII between QA and QB quinone binding
sites, namely, QAFe2+
QB [56,57], at the catalytic Mn cluster of the
oxygen evolving complex (OEC) [58] and at the YD tyrosine residue
of D2 protein [59]. The proposed target sites of NO binding are
illustrated in Fig. 1.
36 A.N. Misra et al. / Nitric Oxide 39 (2014) 35–45](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)