Journal of Experimental Botany, Vol. 59, No. 1, pp. 25–35, 2008
Transport of Plant Growth Regulators Special Issue
doi:10.1093/jxb/erm218 Advance Access publication 1 November, 2007
SPECIAL ISSUE REVIEW PAPER
Nitric oxide evolution and perception
Steven Neill1,*, Jo Bright1, Radhika Desikan2, John Hancock1, Judith Harrison1and Ian Wilson1
1Centre for Research in Plant Science, Faculty of Applied Sciences, University of the West of England, Bristol,
Bristol BS16 1Q, UK
2Division of Biology, Imperial College London, London SW7 2AZ, UK
Received 14 June 2007; Revised 24 July 2007; Accepted 1 August 2007
Various experimental data indicate signalling roles for
nitric oxide (NO) in processes such as xylogenesis,
programmed cell death, pathogen defence, flowering,
stomatal closure, and gravitropism. However, it still
remains unclear how NO is synthesized. Nitric oxide
synthase-like activity has been measured in various
plant extracts, NO can be generated from nitrite via
nitrate reductase and other mechanisms of NO gener-
ation are also likely to exist. NO removal mechanisms,
for example, by reaction with haemoglobins, have also
been identified. NO is a gas emitted by plants, with the
rate of evolution increasing under conditions such as
pathogen challenge or hypoxia. However, exactly how
NO evolution relates to its bioactivity in planta remains
to be established. NO has both aqueous and lipid
solubility, but is relatively reactive and easily oxidized
to other nitrogen oxides. It reacts with superoxide to
form peroxynitrite, with other cellular components
such as transition metals and haem-containing pro-
teins and with thiol groups to form S-nitrosothiols.
Thus, diffusion of NO within the plant may be relatively
restricted and there might exist ‘NO hot-spots’ depend-
ing on the sites of NO generation and the local
biochemical micro-environment. Alternatively, it is
possible that NO is transported as chemical precur-
sors such as nitrite or as nitrosothiols that might
function as NO reservoirs. Cellular perception of NO
may occur through its reaction with biologically active
molecules that could function as ‘NO-sensors’. These
might include either haem-containing proteins such as
guanylyl cyclase which generates the second messen-
ger cGMP or other proteins containing exposed re-
active thiol groups. Protein S-nitrosylation alters
protein conformation, is reversible and thus, is likely
to be of biological significance.
Key words: Arginine, cyclic GMP, GSNO, haem, nitric oxide,
nitrite, perception, peroxynitrite, S-nitrosylation, S-nitrosothiol,
superoxide, transport, tyrosine nitration.
In recent years nitric oxide (NO) has emerged as an
important endogenous signalling molecule in plants that
mediates many developmental and physiological pro-
cesses including xylogenesis, programmed cell death,
pathogen defence, flowering, stomatal closure, and gravi-
tropism (Lamattina et al., 2003; Neill et al., 2003;
Delledonne, 2005; Lamotte et al., 2005). Experimental
evidence in support of such signalling roles for NO has
typically been obtained via the application of either NO or
NO donors (NO itself is a reactive gas with a short half-
life in air), via the measurement of endogenous NO and
through the manipulation of endogenous NO content by
chemical and genetic means. There are potential compli-
cations with using NO donors (Floryszak-Wieczorek
et al., 2006) and undoubtedly technical problems associ-
ated with assaying the NO content of and release from
plants (Planchet and Kaiser, 2006a, b). Moreover, in some
situations, NO can be released in far higher amounts than
would probably be required to effect biological responses
which raises the question of how it can actually function
as a biological signal. NO also has paradoxical effects.
For example, it is growth promoting at low concentra-
tions, but quite inhibitory or toxic at high concentrations
(Beligni and Lamattina, 1999) and being reactive, is
perhaps unlikely to travel far between or even within
cells. It may be that in the rush of enthusiasm to ascribe
biological roles to NO some problems have been over-
looked and with hindsight some of the experimental data
may require re-evaluation.
* To whom correspondence should be addressed. E-mail: Steven.Neill@uwe.ac.uk
ª The Author . Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: firstname.lastname@example.org
by guest on June 4, 2013
This short review, part of a series on ‘Transport of plant
growth regulators’, focuses on NO evolution and perception
by plants and inevitably, perhaps, raises more questions
NO generation and removal in plants
It is clearly important to elucidate the mechanisms by
which NO is biosynthesized in plant cells. However,
despite all the research effort over the last 10 years or so,
there is still much uncertainty. Most work has focused on
two potential enzymatic sources of NO in plants, nitric
oxide synthase (NOS) and nitrate reductase (NR), but
recent research has also alluded to other potential sources
of NO in different compartments of plant cells (Fig. 1A).
Prior to the complete sequencing of the Arabidopsis
genome, early work on NO signalling in plants used
pharmacological inhibitors of NO-generating enzymes to
indicate the potential source of NO. In addition to non-
specific inhibitors of NR, these inhibitors included com-
pounds such as NG-nitro-L-arginine methyl ester (L-NAME)
and NG-monomethyl-L-arginine acetate (L-NMMA), ana-
logues of arginine expected to function as competitive
inhibitors of NOS. Tungstate, that probably replaces moly-
bdenum in the NR enzyme, has also been used as a
potential NR inhibitor. The inhibition of physiological re-
sponses such as programmed cell death, stomatal closure or
root growth by these compounds suggested that either NOS
or NR were likely to be sources of endogenous NO (Neill
et al., 2003). Early work by del Rio and colleagues (del Rio
et al., 2002) used immunogold labelling to indicate that
NOS-like enzymes were present in pea peroxisomes, but
the cloning of a pea homologue of NOS was not reported.
In furthering this work, the peroxisomal NOS activity was
biochemically characterized and arginine-dependent NO ac-
cumulation measured by chemiluminescence and electron
paramagnetic resonance (EPR) spectroscopy (Corpas et al.,
2004a). In addition, constitutive NOS activity, which ap-
peared to be developmentally regulated, was detected in the
leaves, stems, and roots of pea seedlings (Corpas et al.,
2006). More recently, arginine-dependent, salinity-induced
increases in NOS activity have also been demonstrated in
olive (Valderrama et al., 2007). What still remains to be
achieved, however, is the identification of genes encoding
the enzymes responsible for these activities.
The first genetic evidence for a NOS-like enzyme in
plants came from the work of Crawford and colleagues who
identified an Arabidopsis orthologue of mammalian NOS,
named AtNOS1 (Guo et al., 2003). The encoded protein,
AtNOS1, had similarity to one from a snail that was
possibly involved in NO synthesis. Importantly, AtNOS1
was shown to possess the biochemical characteristics of
NOS in that it reduced arginine to citrulline when assayed
with a commercial NOS-assay kit. More importantly still,
a T-DNA insertion mutant, Atnos1, was identified that
produced much reduced levels of NO in guard cells and
roots in response to ABA. Collectively, these data strongly
suggested that AtNOS1 was truly a source of NO in
Arabidopsis. Indeed, other studies also confirmed that the
mutant, Atnos1, was deficient in NO synthesis and action
(He et al., 2004; Zeidler et al., 2004; Bright et al., 2006).
However, the function of AtNOS1 as a NOS has now
been seriously questioned, with three parallel reports in
2006 discussing the problems associated with this con-
cept. It now appears that AtNOS1 may not actually be
a NOS at all. Certainly, it has been difficult reproducibly
to demonstrate typical NOS activity. Various researchers
have been unable to reproduce the results of the earlier
work and detect citrulline when using the arginine-to-
citrulline conversion kit and working with either AtNOS1
or related enzymes from other species (Zemojtel et al.,
2006). Other tests to detect NO arising from the activity of
this enzyme have also failed (Crawford et al., 2006). The
current view is that, although AtNOS1 may not be a NOS
per se, it is somehow involved in NO synthesis or
Fig. 1. NO generation (A) and removal B) in plant cells. NO can be
generated from arginine by NO synthase enzymes; AtNOA1 is required
for this process in Arabidopsis. NO can also be produced from nitrite
2) by nitrate reductase (NR), by nitrite:NO reductase (Ni-NOR) and
via mechanisms yet to be fully defined in chloroplasts and mitochon-
dria. NO reacts readily with oxygen and superoxide. NO is metabolized
by reaction with haemoglobins and reacts with glutathione to form S-
nitrosoglutathione (GSNO). GSNO is catabolized by GSNO reductase.
26 Neill et al.
by guest on June 4, 2013
accumulation. Hence, a name change to Arabidopsis
thaliana Nitric Oxide Associated 1 (AtNOA1) has been
suggested (Crawford et al., 2006). AtNOS1, or as we shall
now call it, AtNOA1, has a conserved GTPase domain
and because it is probably targeted to the mitochondria
(Guo and Crawford, 2005), it has been speculated that it
may be a GTPase involved in mitochondrial ribosome
biogenesis. Presumably impaired AtNOA1 activity would
then be expected to result in impaired mitochondrial
function and thus, altered NO levels (Zemojtel et al.,
2006). However, GTPase activity is yet to be shown for
AtNOA1. Moreover, Guo (2006) argues that the lack of
detection of NOS activity from AtNOA1 and its homo-
logues could be due to the fact that their NAD(P)H-
dependent activity may be very low compared with that of
their mammalian counterparts. It was also suggested that
AtNOA1 may use the stable NO synthesis intermediate,
N-w-hydroxyarginine (NOHA), rather than arginine to
produce NO. Such an intermediate would not be detect-
able by the traditional NOS assay. In addition, it has been
suggested that there may be other co-factors yet to be
identified in plants which could be required to regulate
ATNOA1 activity (Guo, 2006).
Realistically, these recent developments lead us back to
square one: no plant NOS gene has yet been identified.
They also highlight a major caveat in the field of NO
research in that the traditional NOS assay kits that have
been used for mammalian enzymes may not be appropri-
ate for plants. Additional methods such as EPR spectros-
copy and chemiluminesence to detect arginine-dependent
NOS activity would be suitable alternatives (Corpas et al.,
2004a). Nevertheless, the substantial pharmacological data
resulting from the use of NOS inhibitors to inhibit
biological responses and NO production do indicate that
there must be enzymes in plants that are thus affected. By
definition, these enzymes are NOS enzymes, but how they
use arginine to make NO remains to be seen. It is not un-
likely that a plant NOS will have little sequence similarity
with its mammalian counterpart, but will still contain
domains which allow its redox functions to occur. Such
domains could even be located on different polypeptides
which could be brought together following a signalling
event. If such a scenario were to be correct, the identifica-
tion of the NO-producing enzyme in plants could be more
difficult than previously thought, but one may assume that
the enzyme would have a redox function and contain bind-
ing sites for redox prosthetic groups such as flavin or haem.
Another enzymatic source of NO is NR. The primary
function of the NR family of enzymes in plants is one of
nitrogen assimilation by converting nitrate to nitrite.
However, NR can also convert nitrite to NO via a
NAD(P)H-dependent reaction. This was shown originally
in vivo using mutant NR soybean plants (Dean and
Harper, 1986), but has also been shown in vitro using
purified NR and plant extracts (Rockel et al., 2002; Neill
et al., 2003). The first genetic evidence of a physiological
role for the generation of NO by NR was in ABA-induced
stomatal closure in Arabidopsis (Desikan et al., 2002). NR
uses nitrite as a substrate to generate NO and we have
been able to show this for Arabidopsis NR in vitro and in
vivo (Bright et al., 2006). The inhibition of NR activity
with tungstate inhibited both ABA and nitrite-induced
stomatal closure and prevented NO generation (Desikan
et al., 2002; Bright et al., 2006), thus implying that a NR-
like enzyme did play a role in generating the NO normally
required to produce these responses. Importantly, it has
also been possible to show the inhibition of purified NR
activity in vitro using tungstate (Bright et al., 2006).
Arabidopsis contains two NR genes, NIA1 and NIA2,
which have a high degree of coding sequence similarity
and result in two isoforms which are 83.5% identical at
the amino acid level, but which show some localized areas
of sequence divergence in the first 90 N-terminal amino
acids and in various other regions within the two proteins.
Use of the Arabidopsis nia1nia2 NR double mutant
confirmed a role for one or both of the encoded enzymes
in guard cell responses to ABA (Desikan et al., 2002).
The observation that nitrite did not induce NO generation
in nia1nia2 guard cells suggests that this requirement for
NR reflected its in vivo capacity to produce NO from
nitrite. However, as pointed out by Crawford (2006), a
lack of NR may have several effects on plant N metabolism
and indeed Modolo et al. (2006) have reported that the
arginine content of nia1nia2 leaves is substantially reduced.
Our recent work using single NR mutants indicates that
NIA1, which is usually present at a much lower abundance
than NIA2, is the source of NO during ABA signalling
(Bright et al., 2006). These data also suggest that the
aberrant NO biology in nia1nia2 is due specifically to the
lack of NIA1 as opposed to generally aberrant N metabo-
lism. Interestingly, Yu et al. (1998) similarly concluded
that NIA1 and NIA2 have distinct signal transduction and
nitrogen assimilatory roles. Key questions must then relate
to the differential expression of their encoding genes, their
subcellular localization and interacting protein partners,
their activation characteristics and the functional signifi-
cance of their partial sequence divergence. NO generation
by NR is stimulated by hypoxic conditions and in spinach
and maize NR-mediated NO generation can be modulated
by the phosphorylation status of the NR (Rockel et al.,
2002). Thus, a potential regulatory mechanism may exist in
vivo. Increasing endogenous nitrite concentrations, either
by dark treatment or by antisense-inhibition of endogenous
nitrite reductase activity (Morot-Gaudry et al., 2002;
Rockel et al., 2002), increases NO emission. NR-mediated
NO generation has also been demonstrated in roots with
a potential physiological role, that of mediating aerenchyma
formation, having been suggested (Dordas et al., 2003).
As shown in tobacco, mitochondrial reduction of nitrite
to NO can also be a major source of NO with tissue nitrite
Nitric acid evolution and perception 27
by guest on June 4, 2013
concentrations being a major limiting factor and NR
function obligatory (Planchet et al., 2005). However, it is
not clear whether or not this occurs in both leaves and
roots (Gupta et al., 2005; Modolo et al., 2005). In
addition, soybean chloroplasts have recently been identi-
fied as a source of NO via arginine or nitrite (Jasid et al.,
2006). However, in this latter case, the enzymes regulat-
ing both arginine and nitrite-dependent NO formation are
not yet known. Apoplastic, non-enzymatic conversion of
nitrite to NO at low pH has also been demonstrated in the
barley aleurone layer (Bethke et al., 2004).
A plasma membrane-bound, root-specific enzyme, ni-
trite-NO oxidoreductase (Ni-NOR), may also function as
a further source of NO. This enzyme was identified
biochemically via its NO-generating activity. However,
unlike NR, it does not use NAD(P)H as a cofactor, but
uses cytochrome c as an electron donor in vitro and has
a comparatively reduced pH optimum. However, neither
its physiological role nor its genetic identity is yet known
(Stohr and Stremlau, 2006).
Other enzymes may also be involved in NO production
(Corpas et al., 2004b). For example, in animals, xanthine
oxidoreductase (XOR), under hypoxic conditions, can
produce NO in preference to H2O2(Millar et al., 1998).
However, Planchet and Kaiser (2006b) were unable to
observe any NO production from recombinant xanthine
oxidase. Interestingly, Arnaud et al. (2006) demonstrated
a plastid-located, iron-induced NO burst in Arabidopsis
that, although susceptible to inhibition by L-NAME,
required neither AtNOS1 nor NR. Such novel NO sources
Removal of nitric oxide
It is likely that biologically active molecules such as NO
are rapidly removed or metabolized following initial
signalling events. It is also possible that increased rates of
NO accumulation or emission actually reflect reduced
rates of removal rather than increased generation. Thus,
the importance of determining how NO levels are
controlled is of obvious importance (Fig. 1B). Simple
chemical reactions are often responsible for the removal of
NO from solution. Nitric oxide is inherently unstable and
will readily react with oxygen to form nitrite and nitrate.
As described above, nitrite can act as a precursor to NO
and may have some biological activity per se (Gladwin
et al., 2005).
The free radical nature of NO means that it will readily
react with other radicals that might also be present. In
both animals and plants, NO is often produced at the same
time and in the same place as Reactive Oxygen Species
(ROS) such as superoxide anions. Superoxide and NO
will react in a stoichiometric manner to produce peroxy-
nitrite (ONOO–). Although it has been noted that plant
cells, unlike animal cells, appear resistant to peroxynitrite
(Delledonne et al., 2001), it may have intrinsic signalling
properties. Whether or not this turns out to be the case, the
level of NO can be instrumental in controlling ROS levels
in cells and vice versa. It has been noted that the basal
rates of NO production in leaves are often under-estimated
because the NO reacts rapidly with superoxide anions
(Vanin et al., 2004).
NO reacts readily and reversibly with either thiol groups
in the cysteine residues of proteins or with the tripeptide
glutathione (GSH) and protein S-nitrosylation may be a
key facet of NO signalling (see below). Glutathione con-
centrations are typically 2–3 mM in plant cells (Ball et al.,
2004) and thus, formation of S-nitrosylated glutathione
(GSNO) could have a large impact on the concentration of
free NO. GSNO is metabolized by the enzyme GSNO
reductase (Diaz et al., 2003; Fig. 1B) and this enzyme
may be instrumental in controlling the bioavailability of
NO and the formation of protein S-NO groups, thereby
regulating such NO-regulated processes as, for example,
plant pathogen defence responses (Feechan et al., 2005).
As well as reacting with thiol groups, NO can also in-
teract with transition metals, particularly with iron which
is often associated with haem as in guanylyl cyclase (see
below) and it has long been recognized that NO can react
with haemoglobins. Non-symbiotic haemoglobins (nsHbs)
from barley, alfalfa, and Arabidopsis are known to react
with NO resulting in its removal from solution. Nitrate is
formed in a NAD(P)H-dependent reaction with the ox-
idized haem intermediate being re-reduced by either the
NAD(P)H or FADH or, as in the case of the barley
haemoglobin, by methaemoglobin reductase (reviewed by
Perazzolli et al., 2006). Arabidopsis AHb1 is also S-
nitrosylated (Perazzolli et al., 2004). Interestingly, nsHbs
are induced by certain treatments where NO generation
might be enhanced, for example, by low partial pressures
of O2(Trevaskis et al., 1997) or by nitrate or nitrite and
by NO itself (Wang et al., 2000; Ohwaki et al., 2005;
Shimoda et al., 2005; Sasakura et al., 2006). Transgenic
manipulation of AHb1 affects NO evolution, which, cor-
related with the ability to survive hypoxic stress
(Perazzolli et al., 2004), indicates a physiological role for
AHb1 in modulating NO levels. Further evidence for the
endogenous NO-detoxifying action of Hb comes from
work involving plant–microbe interactions. Boccara et al.
(2005) showed that HmpX, an Erwinia chrysanthemi
flavohaemoglobin and virulence determinant, removed NO.
Infection with a HmpX-deficient mutant of E. chrysanthemi
triggered high levels of NO coupled to the hypersensitive
response in the host plant. Sasakura et al. (2006) showed
that a nsHb in the actinorhizal plant Alnus firma is highly
expressed in nodules and may serve to detoxify NO.
Although there is no doubt that plants perceive and
respond to NO, the mechanisms by which such perception
28 Neill et al.
by guest on June 4, 2013
occurs still require clarification. There is now considerable
research interest concerning this question, but as no
specific plant NO receptor has been identified, work in
this area has taken its lead from mammalian research. The
reactive nature of NO and its ability to interact with and
modify many proteins suggests that there may turn out to
be many ‘NO perceptors’ (Fig. 2). In animal cells, soluble
guanylyl cyclase (sGC) has a key role in NO signalling.
NO activates sGC by binding to its haem domain stimulat-
ing a transient rise in cGMP levels which, in turn, ac-
tivates a number of targets. In plants, pharmacological
studies using inhibitors of NO sensitive guanylyl cyclase
have implicated cGMP downstream of NO and ABA
signalling in guard cells (Neill et al., 2003). NO induces
an increase in cGMP (Durner et al., 1998) and work in
our laboratory has shown that application of ABA or the
NO donor SNP to guard cell-enriched preparations from
Arabidopsis induces a small and transient increase in
cGMP that can be prevented by the application of the GC
(ODQ) or the NO scavenger PTIO (J Harrison et al., un-
published data). Thus, a similar mechanism of NO stim-
ulated cGMP synthesis may also operate in plants. A key
signalling molecule downstream of cGMP is cyclic ADP-
ribose (cADPR) (Wendehenne et al., 2001). In animal
cells cADPRstimulates Ca2+release viaintracellular ryano-
dine receptor calcium channels (RYR) and it is possible
that a similar signalling mechanism operates in plants. In
tobacco, cADPR elevates the expression of the genes
encoding phenylalanine ammonia lysase (PAL) and the
pathogenesis-related protein 1 (PR-1) in a manner that is
sensitive to RYR inhibitors (Durner et al., 1998). These
genes are also NO-regulated and cADPR antagonists re-
duce the expression of PR-1 (Klessig et al., 2000). NO is
known to cause increases in the level of free Ca2+(Durner
et al., 1998; Garcia-Mata et al., 2003). Thus, NO may
signal through cGMP, cADPR, and Ca2+to promote its
effects. NO, cGMP, and cADPR have all also been shown
to mediate ABA-induced stomatal closure (Neill et al.,
2003; Garcia-Mata and Lamattina, 2002). The cPTIO in-
hibition during this process of the ABA-induced inactiva-
tion of the Ca2+-dependent inward rectifying K+channel
and activation of the outward rectifying Cl–channel
(Garcia-Mata et al., 2003) strongly implicates NO and
Ca2+in the signalling cascade that may operate.
Mammals, vertebrates, insects, and many lower eukary-
otes possess sGCs with a haem domain capable of
binding NO. Bacterial sGCs contain a similar NO binding
domain termed the H-NOX domain (Karow et al., 2004;
Boon et al., 2006). However, plant homologues of the
animal NO sensitive sGC have yet to be identified. The
Arabidopsis guanylyl cyclase, AtGC1, is apparently not
activated by NO (Ludidi and Gehring, 2003). Thus, the
question remains as to how and by what signalling pro-
cess NO induces a rise in the level of cGMP in plants and
it may be that plant enzymes that generate cGMP in
response to this gas are quite different from their mam-
malian counterparts. Indeed, a recent report has demon-
strated that the Arabidopsis brassinosteroid receptor BRI1
contains a domain with guanylyl cyclase activity (Kwezi
et al., 2007) indicating that there may well be more novel
plant guanylyl cyclases awaiting discovery.
The redox chemistry of NO facilitates its reaction with
iron–sulphur and haem groups which are present in a
number of different proteins. In addition, NO may also
signal its presence through other mechanisms such as
either direct S-nitrosylation or indirect trans-nitrosylation
of either protein cysteine residues or low molecular
weight compounds such as glutathione or via peroxynitrite
nitration of tyrosine residues (Fig. 2; see Mur et al., 2006,
for an excellent discussion of NO chemistry). In animals,
S-nitrosylation has been shown to regulate a number of
signalling processes, stuctural proteins, and metabolic
pathways and has become established as the prototype
redox-based, post-translational protein modification in the
animal kingdom (Wang et al., 2006). In plants, evidence
is now beginning to emerge that S-nitrosylation may also
play an important role in NO signalling. A number of
proteins appeared to become S-nitrosylated when extracts
of Arabidopsis cell cultures were treated with GSNO and
SNO-containing proteins isolated by the biotin switch
method (Lindermayr et al., 2005). The proteins identified
were involved in a wide range of cellular processes.
However, their in vivo S-nitrosylation and its biological
significance remain to be seen. The in vitro activity of one
of three recombinant methionine adenosyl transferase
Fig. 2. Potential mechanisms of NO perception. NO may be perceived
in plants by a number of mechanisms that differ depending on the cell
type, intracellular location, biochemical microenvironment, and envi-
ronmental stimuli. NO can bind to the haem domain in proteins such as
guanylate cyclase and with metals to form metal-nitrosyl complexes. It
can also react with the SH group of low molecular weight thiols such as
glutathione to form S-nitrososglutathione (GSNO) and, either directly
or via GSNO, nitrosylate proteins to form S-nitrosylated proteins.
S-nitrosylation induces conformational changes and is reversible.
NO reacts with superoxide to form peroxynitrite which can then nitrate
proteins on tyrosine residues. It is not yet known whether this reaction
has signalling consequences.
Nitric acid evolution and perception 29
by guest on June 4, 2013
isoforms has been shown to be altered by S-nitrosylation
in a manner dependent on the presence or absence of Cys-
114 (Lindermayr et al., 2006). Similarly, the activity of an
Arabidopsis metacaspase appears to be dependent on the
nitrosylation of a critical cysteine residue (Belenghi et al.,
2007). Under lowered partial pressures of O2 the
mammalian RyR1 calcium channel also becomes S-
nitrosylated on a specific Cys residue at physiologically
relevant NO levels and in a manner dependent on the
presence of calmodulin (Eu et al., 2000). Should this
occur in plants, it would have obvious relevance in terms
of NO signalling and responses and there is some work
suggesting that this may be the case in stomatal guard
cells (Sokolovski and Blatt, 2004).
As protein S-nitrosylation can be mediated by GSNO,
formed by the S-nitrosylation of GSH (Wang et al., 2006),
the degree of protein S-nitrosylation and thus, ‘NO
activity’, will be reflected in the availability of reactive
GSNO. An Arabidopsis GSNO reductase, AtGSNOR1,
has now been identified and its biological importance
highlighted (Sakamoto et al., 2002; Diaz et al., 2003;
Feechan et al., 2005). Loss-of-function mutations of this
gene increased S-nitrosylation levels and disabled R
(Resistance)-gene related defence responses against mi-
crobial pathogens (Feechan et al., 2005). Conversely,
gain-of-function mutants were enhanced in their defensive
ability. It was demonstrated that AtGSNOR1 positively
regulated the signalling network controlled by the plant
immune system activator, salicylic acid. In pea, both
GSNO reductase activity and gene expression are de-
creased by cadmium stress (Barroso et al., 2006). Thus,
there is definitely a case for S-nitrosylation being involved
in signalling pathways which may include that for NO.
Obviously the study of S-nitrosylation in plants is in its
infancy and much work is required to determine on which
specific proteins it occurs in vivo during the different
physiological processes regulated by NO. Various protein
S-nitrosylation motifs have also been suggested, based on
the appropriate regions of animal proteins that are known
to be affected. Wang et al. (2006) suggested the motif
[HKR]-C-[VILMFWC]-x-[DE] as that targeted by NO
and the motif [GSTCYNQ]-[KRHDE]-C-[DE] has been
suggested as that targeted by GSNO. Scanning the
Arabidopsis protein databases with these motifs yields
231 and 241 hits, respectively. While the proteins
identified include a number of MAP kinases and other
signalling proteins, none of those identified as being
S-nitrosylated by Lindermayer et al. (2005) are present in
the lists of proteins generated. Thus, there is probably
no substitute for laboratory-based investigations in this
case. However, a number of potential, bioinformatically-
generated targets could be examined in transgenic mutant
complementation experiments where the highlighted Cys
is either present or absent for nitrosylation in the com-
It is also possible that NO signals via the nitration of
tyrosine residues. Tyrosine nitration is mediated by
reactive nitrogen species such as the peroxynitrite anion
(ONOO–) and nitrogen dioxide (NO2) which are formed
during the metabolism of NO in the presence of oxidants
such as superoxide radicals (O?
(H2O2), and transition metal centres (Radi, 2004). Al-
though the peroxynitrite anion can cause tyrosine nitration
in vitro, its role in this process has been questioned and
alternative mechanisms have come to the fore that depend
on the formation of NO2 by the action of haem
peroxidases on nitrite (Brennan et al., 2002). A number
of recombinant Arabidopsis haemoglobins that exhibit
peroxidase-like activity and differentially mediate nitrite-
dependent protein nitration in vitro have been identified
(Sakamoto et al., 2004). Endogenous protein tyrosine
nitration has also been demonstrated, in mutant tobacco
plants with greatly increased amounts of NO (Morot-
Gaudry et al., 2002) and more recently in olive leaves
where the amount of tyrosine nitrated proteins increased
under salt stress (Valderrama et al., 2007). Thus, the
extent and biological significance of protein nitration and
whether or not what appears to be a non-reversible
reaction can act as a signalling process, presumably in
tandem with protein turnover, remains to be determined.
2), hydrogen peroxide
It is possible that NO can diffuse within a cell from
a specific site of generation, say in the mitochondria, to
other regions of the cell where it might induce an effect by
interaction with specific target proteins. It is also possible
that NO can diffuse out of the cell across the plasma
membrane into adjacent cells and thereby create a small
region of cells responding to NO. However, whether or
not NO does diffuse within and between cells and if it
does how far it moves remains unknown. Given that cells
clearly contain many proteins and other molecules that
react with NO, it might be that such diffusion is limited.
This could of course be the case unless the NO
concentration were to be sufficiently high, not necessarily
across the whole cell, but perhaps in a microlocale within
the cell, so as to saturate, transiently at least, such ‘NO-
binding molecules’ in its immediate vicinity. This would
leave non-reacted NO free to diffuse across and out of
cells. It is likely that cellular regions do have higher local
NO concentrations either because they contain the bio-
chemical machinery required for NO synthesis or because
NO accumulates preferentially in such regions. For
example, NO is more soluble in lipid than water and so
may accumulate preferentially in membranes where its
rates of reaction with any interacting molecules may be
consequently higher (Liu et al., 1998). Different stimuli
may activate NO synthesis either by different mechanisms
30 Neill et al.
by guest on June 4, 2013
and/or in different subcellular compartments and there
may also be a directional focus. For example, a bacterium
or fungal hypha may abut only one region of a plant cell
and the resultant signalling might activate NO generation
in only a proximal and discrete region of the cell. If NO-
response proteins such as ion channels or second
messenger-generating enzymes are also co-located then
one could envisage local ‘NO hot-spots’ and NO signal-
ling micro-domains (Fig. 3). Although it may well be
technically difficult to monitor NO transport, it may be
informative to apply NO via a point source to the exterior
or interior of a tissue and then to monitor real-time NO
movement by, for instance, fluorescent imaging using
a NO-sensing dye such as DAF-2DA.
An alternative, but not exclusive scenario for NO
transport might be that its generation is elevated in
discrete regions owing to localized stimulation resulting
from the long-distance transport and site-specific accumu-
lation of compounds such as the hormones ABA or IAA
that can stimulate its production. Directional transport of
IAA is well-known, particularly with respect to its role in
mediating tropic responses to gravity and light. NO has
been implicated in gravity signalling with localized NO
accumulation being induced either by gravistimulation or
asymmetric IAA application and prevented by the in-
hibition of IAA transport (Hu et al., 2005). The systemic
transport of ‘defence-signals’ is activated by pathogen
challenge and it may be that these signals also stimulate
NO generation at sites distant from those of the initial
Another possibility awaiting clarification is that NO
precursors or ‘NO storage compounds’ may be trans-
ported with either NO generation or release occurring at
distant sites in a manner analogous to the transport of the
ethylene precursor ACC. GSNO has been suggested as
one transportable form of NO and although it has not yet
been unequivocally identified in plants, a recent report
demonstrated cross-reactivity with an anti-GSNO antibody
in pea collenchyma cells and immunofluorescence micros-
copy indicated that the GSNO content decreased dramat-
ically under cadmium stress (Barroso et al., 2006). GSNO
has recently been demonstrated in leaf vascular tissue and
shown to increase under salt stress using confocal laser
microscopy (Valderrama et al., 2007). Glutathione is also
present at high (e.g. millimolar levels in wheat) concen-
trations in phloem cells (Bourgis et al., 1999). Arginine
and nitrite could also serve as transported NO precursors.
Nitrite concentrations in the phloem and xylem are
unknown. However, whole tissue nitrite concentrations,
which are typically 10–20 lM, can be transiently raised
above this level (Rockel et al., 2002) which may be
indicative of the movement of NO precursors. Arginine
concentrations can be quite high [e.g. 250 lM in
Arabidopsis leaves (Modolo et al., 2006) and 300–800
lM in melon phloem] and, interestingly, can be increased
by ABA treatment (Mitchell and Madore, 1992).
NO evolution from plants
There can be no doubt that NO is evolved from plants.
Such evolution, measured as NOx(a mixture of NO and
NO2), was first reported in the 1980s and shown to be
increased by treatment with salicylic acid and various
other compounds (Harper, 1981; Dean and Harper, 1986;
Klepper,1990). Wildt et al. (1997) measured NO emis-
sions from several species and several other reports have
demonstrated that NO evolution from plants can increase
or decrease in response to treatments such as pathogen
challenge, water stress, exposure to UV-B, the application
of fungicides, and anoxic conditions (Lesham and
Haramaty, 1996; Clarke et al., 2000; Magalhaes et al.,
2000; Rockel et al., 2002; Hari et al., 2003; Conrath et al.,
2004; Perazzolli et al., 2004; Mur et al., 2005, 2006).
However, there are a number of technical and biological
uncertainties with these measurements and quite varied
rates of NO evolution have been estimated using a variety
of different measuring techniques. Of course, it is also
difficult to gauge the biological significance of the NO
evolved. Typically, the rates of evolution are in the nmol
g?1h?1range (Table 1). A key question is whether or not
this NO evolution reflects increased concentrations of
biologically active NO in planta. In some cases, this seems
likely to be the case. For example, during responses to
pathogen challenge there are good correlations between NO
Fig. 3. ‘NO cell map’. NO is made and removed in cells by several
potential mechanisms and at several intracellular localizations. NO
reacts readily with various protein and non-protein partners, potentially
minimizing NO diffusion in, and between cells and into the apoplast
and atmosphere. Thus NO may be localized to potential ‘NO hot-spots’,
cellular microdomains associated with sites of synthesis and action or
Nitric acid evolution and perception 31
by guest on June 4, 2013
evolution and the biological responses that occur. Such cor-
relations have been shown using either NO scavengers and
NO synthesis inhibitors or virulent and avirulent pathogens.
However, as pointed out by Planchet and Kaiser (2006a, b),
estimates of intracellular NO content and rates of NO
evolution do not always agree. For example, during anoxia
NO is generated at a much higher level than is probably
required for its cell signalling function. Such paradoxes
remain to be resolved, but the concept of localized NO
generation and action (Fig. 3) may partly explain them.
It is not known if NO released from one part of a plant
can induce effects on either other parts of the same plant
or on adjacent plants. Agricultural soils can also release
substantial amounts of NO (Davidson and Kingerlee,
1997). Certainly, NO gas does have effects on plant
biology. Indeed, the early work on NO concerned its
effects as an air pollutant (Mansfield, 2002). NO is still an
air pollutant today and plants in urban areas or close to
traffic are likely to receive higher chronic and more acute
exposures than those in rural areas (rural locations 7–70
nl l?1, urban 20–900 nl l?1; Environment Agency, 2006).
The early work showed that NO at 50–500 nl l?1(urban
smog [NO] <5000 nl l?1) could retard growth and inhibit
photosynthesis (Mansfield, 2002) and other studies have
shown that trace amounts of NO in smoke can stimulate
seed germination (Keely and Fotheringham, 1997). This
latter work has recently been questioned (Baldwin et al.,
2005), but physiological concentrations of NO gas do
appear to stimulate seed germination (Bethke et al., 2006)
and very short exposures to high concentrations of NO
gas have substantial effects on the transcriptome (Huang
et al., 2004). Thus, there is much fertile ground for further
research. For instance, does NO released by plants
contribute to the global N economy? Under waterlogged
conditions plant NO evolution may be substantial. Does
NO, after reaction with other atmospheric gases such as
ozone, contribute to the ‘Greenhouse Effect’ and what
effects does atmospheric NO have on plants?
Clearly there is still much to be discovered about NO
synthesis, evolution, and perception in plants. NO is
undoubtedly made by plant cells and has a range of
biological activities. Therefore, it would seem likely that
the processes by which NO is made and removed are
subject to regulation. Even though the details remain to be
resolved, it is clear that various stimuli can increase the
rate of NO production and that altering NO turnover in
cells, either by modulating its production or removal, does
have biological effects. NO is evolved from plants and the
rate of evolution can be dramatically increased in response
to various stimuli, but the physiological significance of
such evolution is not clear. Increased NO evolution
probably reflects increased cellular NO generation, but
whether NO derived from different intracellular sources is
evolved at different rates or from different cells is not
known. NO appears to be generated locally in response to
mobile signals, but again it remains to be seen if either
NO per se acts as a mobile, diffusible signal or if NO
reservoirs or precursors are transported and importantly,
whether or not such transport is regulated. NO reacts with
many other molecules inside and outside of cells. This
includes reactions with oxygen to form nitrogen oxides,
with GSH and proteins during the S-nitrosylation of thiol
residues and with superoxide to form peroxynitrite during
the nitration of tyrosine residues within proteins. Thus,
NO perception may well involve several mechanisms and
it could be that NO is unlikely to travel far even within
a single cell and, consequently, not far between cells. It is
possible that local pathogen or hormone induced ‘NO-
hotspots’ exist within cells and tissues and that the extent
and duration of the accumulation of NO at these sites is
a balance between synthesis and removal. There is clearly
much we do not know.
Work in the authors’ laboratory was supported by BBSRC, the
Leverhulme Trust, and the Wellcome Trust.
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Control; P. syringae-challenged
Control; P. syringae-challenged
Control; P. syringae-challenged
Laser photoacoustic detection
Laser photoacoustic detection
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Perazzolli et al. (2004)
Magalhaes et al. (2000)
Mur et al. (2005)
Mur et al. (2006)
Conrath et al. (2004)
Rockel et al. (2002)
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