Journal of Experimental Botany, Vol. 66, No. 10 pp. 2877–2887, 2015
doi:10.1093/jxb/erv051 Advance Access publication 1 March 2015
REVIEW PAPER
Nitric oxide: a multifaceted regulator of the nitrogen-fixing
symbiosis
Imène Hichri1,2,3, Alexandre Boscari1,2,3, Claude Castella1,2,3, Martina Rovere1,2,3, Alain Puppo1,2,3 and
Renaud Brouquisse1,2,3,*
1 INRA, Institut Sophia Agrobiotech (ISA), UMR 1355, BP 167, 06903, Sophia Antipolis cedex, France
CNRS, Institut Sophia Agrobiotech (ISA), UMR 7254, BP 167, 06903, Sophia Antipolis cedex, France
3 Université Nice Sophia Antipolis, Institut Sophia Agrobiotech (ISA), BP 167, 06903, Sophia Antipolis cedex, France
2 * To whom correspondence should be addressed. E-mail: [email protected]
Received 28 November 2014; Revised 15 January 2015; Accepted 16 January 2015
Abstract
The specific interaction between legumes and Rhizobium-type bacteria leads to the establishment of a symbiotic
relationship characterized by the formation of new differentiated organs named nodules, which provide a niche for
bacterial nitrogen (N2) fixation. In the nodules, bacteria differentiate into bacteroids with the ability to fix atmospheric
N2 via nitrogenase activity. As nitrogenase is strongly inhibited by oxygen, N2 fixation is made possible by the microaerophilic conditions prevailing in the nodules. Increasing evidence has shown the presence of NO during symbiosis,
from early interaction steps between the plant and the bacterial partners to N2-fixing and senescence steps in mature
nodules. Both the plant and the bacterial partners participate in NO synthesis. NO was found to be required for the
optimal establishment of the symbiotic interaction. Transcriptomic analysis at an early stage of the symbiosis showed
that NO is potentially involved in the repression of plant defence reactions, favouring the establishment of the plant–
microbe interaction. In mature nodules, NO was shown to inhibit N2 fixation, but it was also demonstrated to have
a regulatory role in nitrogen metabolism, to play a beneficial metabolic function for the maintenance of the energy
status under hypoxic conditions, and to trigger nodule senescence. The present review provides an overview of NO
sources and multifaceted effects from the early steps of the interaction to the senescence of the nodule, and presents
several approaches which appear to be particularly promising in deciphering the roles of NO in N2-fixing symbioses.
Key words: Hypoxia, legume, nitric oxide, nitrogen fixation, Rhizobium, symbiosis.
Introduction
The symbiotic association between the roots of legumes and
soil Rhizobium-type bacteria results in the formation of a new
differentiated organ, the nodule, with the primary function
of nitrogen (N2) fixation. After extensive recognition and
signalling processes involving plant flavonoids and bacterial lipochito-oligosaccharides, termed ‘nodulation factors’,
bacteria penetrate root hairs via a specific structure, the infection thread, while meristematic activities are initiated in the
root cortical cells to give birth to the nodule (Long, 2001).
Rhizobia divide within the infection thread, which traverses
the root hair and the cortical cells to reach the cortex, where
bacteria are subsequently released into the host cells. In the
Abbreviations: ARA, acetylene-reducing activity; Arg, arginine; CP, cysteine protease; c-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide;
DAF, 4,5 diamino-fluorescein; DAF-2DA, DAF-2diacetate; DAF-FM, 4-amino-5-methylamino-2’,7’-difluorofluorescein; DETA-NO, diethylenetriamine NONOate;
dpi, days post-inoculation; γ-EC, γ-glutamyl cysteine; γ-ECS, γ-glutamyl cysteine synthetase; ERF, ethylene response factor; ETC, electron-transport chain; Gln,
glutamine; Glu, glutamate; GS, glutamine synthetase; GSH, glutathione; GSHS, glutathione synthetase; GSNO, S-nitroso glutathione; Hb, hemoglobin; Hmp, flavohemoprotein; hpi, hours post-inoculation; Lb, leghemoglobin; LbNO, nitrosylleghemoglobin; L-NMMA, NG-monomethyl-L-arginine; L-NNA, Nω-nitro-L-arginine;
NH4+, ammonium; Nif, nitrogenase; NirK, nitrite reductase K; NO, nitric oxide; NO3–, nitrate; NO2–, nitrite; NOS, NO synthase; N2O, nitrous oxide; NR, nitrate reductase; PAOx, polyamine oxidase; PEP, phosphoenolpyruvate; SNP, sodium nitroprussiate; Tg, tungstate; wpi, weeks post-inoculation.
© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
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2878 | Hichri et al.
nodule, bacteria differentiate into bacteroids, which have the
ability to fix atmospheric N2 via nitrogenase activity (Oldroyd
and Downie, 2008). Nodules may be either determinate or
indeterminate (Hirsch, 1992). Indeterminate nodules, such
as those of pea, clover, and alfalfa, are cylinder shaped,
have a persistent meristem, and comprise four distinct zones
(Fig. 1): zone I is made of meristematic cells; zone II is where
cells are infected by bacteria, which differentiate into bacteroids; zone III is where bacteroids reduce N2 into ammonia,
which is exported to the plant; and zone IV is characterized
by the disruption of the partnership and the onset of senescence (Timmers et al., 2000). Determinate nodules, such as
those of bean, soybean or cow pea, are round shaped, lack a
persistent meristem, and grow through cell expansion rather
than cell division. A central zone hosts infected and uninfected cells where N2 fixation occurs (Franssen et al., 1992).
Legume–rhizobium symbiosis is beneficial for both partners
since, in exchange for reduced nitrogen, the bacterial partner
is supplied with energy and carbon nutrients in the form of
dicarboxylic acids by the plant partner (Lodwig et al., 2003).
N2 reduction by the bacteroid nitrogenase constitutes the
core reaction of the symbiotic process (Oldroyd and Downie,
2008). As nitrogenase is irreversibly inhibited by oxygen
(O2) traces, N2-fixation requires the microaerophilic conditions prevailing in the nodules (Appleby, 1992). Thus, nodule
development occurs in changing conditions, shifting from a
normoxic environment during the establishment of the symbiosis to a microoxic one in functioning nodules.
Nitric oxide (NO) is a free radical, reactive gaseous molecule with a broad spectrum of regulatory functions in
plant growth and development (Besson-Bard et al., 2008),
and responses to biotic (pathogenic and symbiotic interactions) and abiotic stresses (Besson-Bard et al., 2008; Moreau
et al., 2010), including hypoxia (Igamberdiev and Hill, 2009;
Blokhina and Fagerstedt, 2010). Increasing evidence of the
occurrence of NO during legume–rhizobium symbiosis has
been reported during the past decade. Using the cell-permeable NO-fluorescent probe 4-amino-5-methylamino-2’,7’difluorofluorescein (DAF-FM), NO production was shown
to be transiently induced in the roots of Lotus japonicus and
Medicago sativa 4 h post-inoculation (hpi) with their cognate
symbionts (Nagata et al., 2008). At a later stage of the symbiotic interaction, 4 days post-infection (dpi), using both a
specific NO-biosensor Sinorhizobium meliloti strain and the
fluorescent probe DAF-2 diacetate (DAF-2DA), NO was
shown to be produced at different sites during the infection
process: in shepherd’s crooks of root hairs, infection threads,
and nodule primordia of M. truncatula roots (del Giudice
et al., 2011). The first evidence of NO occurrence in N2-fixing
nodules came from the detection of NO complexed to leghemoglobin (Lb), the major hemoprotein of legume nodules. The
presence of such nitrosylleghemoglobin (LbNO) complexes
was reported in several legumes representing determinate and
indeterminate nodule types (Maskall et al., 1977; Kanayama
and Yamamoto, 1991; Mathieu et al., 1998; Meakin et al.,
2007; Sanchez et al., 2010). Using DAF-2DA, Baudouin
et al. (2006) showed that in M. truncatula–S. meliloti nodules NO detection was associated with the N2-fixing zone, but
not with meristematic, infection, and senescence zones. More
recently, using an NO-biosensor S. meliloti strain, Cam et al.
(2012) provided arguments that in M. truncatula nodules, NO
could be produced at interzone III–IV, between N2-fixing and
senescence zones, in the late phase of the symbiosis process.
Considered together, these observations mean that NO is produced throughout the whole symbiotic process (Fig. 1), and
the question was raised as to the origin of NO and its physiological roles in the different time and space of the symbiotic
interaction.
Both the plant and the bacterial partners should be considered as potential sources of NO. In plants, beside a nonenzymatic conversion of NO2– (nitrite) to NO in the apoplast
(Bethke et al., 2004), seven enzymatic pathways for NO
production have been described (Gupta et al., 2011). In the
reductive pathways, NO2– can be reduced to NO through the
action of either nitrate reductase (NR), plasma membranebound nitrite:NO reductase, xanthine oxido-reductase, or
the mitochondrial electron-transport chain (ETC), particularly in a low-O2 environment (Gupta et al., 2011; Mur
et al., 2013). Oxidative pathways that lead to NO production
depend on either arginine, polyamines or hydroxylamine as
primary substrates. This oxidative NO production, mediated
by still uncharacterized enzymes [NO synthase (NOS)-like,
Fig. 1. NO production sites at different steps of the legume–Rhizobium symbiosis. NO production (blue stars) is induced 4 h post-inoculation (1) and
during each step of the infection process: in the shepherd’s crook of the root hair (2), infection thread (3), and nodule primordia (4). In the mature nodule
(5), NO detection is associated with the infection zone, N2-fixing zone, and inter-zone III–IV. wpi, weeks post-inoculation.
Nitric oxide in N2-fixing symbiosis | 2879
polyamine oxidase (PAOx)], occurs under normoxic conditions (Gupta et al., 2011; Mur et al., 2013). In bacteria, the
main route for NO production is the denitrification pathway,
which allows bacteria to respire in an anaerobic environment
(Zumft, 1997), but NO may also be produced in normoxic
conditions by NOS homologues (Sudhamsu and Crane,
2009). Thus, in both plant and bacterial partners, the O2 environment strongly influences NO sources and should be taken
into account when analysing the role of NO in a physiological
context.
NO sources and cellular functions have been extensively
reviewed for both plants (Besson-Bard et al., 2008; Moreau
et al., 2010; Gupta et al., 2011; Mur et al., 2013) and symbiotic bacteria (Sudhamsu and Crane, 2009; Meilhoc et al.,
2011). The present review first provides an overview of NO
sources and multifaceted effects during the N2-fixing symbiosis, from the early steps of the interaction to the senescence of
the nodule. It goes on to present several particularly promising lines of investigation for deciphering the roles of NO in
N2-fixing symbioses.
NO during the establishment of the
symbiosis
The source of NO in the establishment of N2-fixing symbiosis is still unclear. Two main ways have been described as
potential NO-generating systems during the first steps of
the symbiotic interaction: NOS-like (Leach et al., 2010) and
NR (Boscari et al., 2013a). The NOS inhibitor N-nitro-Larginine (L-NNA) negatively affects the interaction of soybean with Bradyrhizobium japonicum (Leach et al., 2010),
suggesting that a NOS-like enzyme is operating and required
for optimal nodulation efficiency. To date, no NOS-like gene
has been identified in plants, but a Nitric Oxide-Associated
gene, named AtNOA1, and encoding a GTPase, was characterized in Arabidopsis (Crawford et al., 2006; Moreau et al.,
2008). The potential involvement of the AtNOA1 orthologue
from M. truncatula, MtNOA1, was evaluated in the symbiotic interaction with S. meliloti (Pauly et al., 2010). Although
MtNOA1 affects the establishment and functioning of the
symbiotic interaction, it does not influence NO production in
the nodules (Pauly et al., 2010). The reductive pathway using
NR is the best characterized source of NO in plants, but
the involvement of this pathway in NO synthesis observed
during the early steps of symbiotic interaction has not yet
been defined. Recent work showed that during early interaction between M. truncatula and S. meliloti, root treatment
with tungstate (Tg), an NR inhibitor, mimics the effects of
the NO-scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) on the transcriptional
regulation of genes implicated in the nodulation process;
however, treatment with the NOS inhibitor L-NAME was
ineffective (Boscari et al., 2013a). These observations support
the probable involvement of NR in the synthesis of NO at
this stage.
Inoculation of legumes (L. japonicus and M. sativa)
with their symbiont (Mesorhizobium loti and S. meliloti,
respectively) induces transient NO production, observed 4
hpi, whereas inoculation with non-symbiotic rhizobia did
not, indicating that NO production results from the specific
recognition of the plant and bacterial partners (Nagata et al.,
2008). Using L. japonicus, Uchiumi’s team demonstrated
that NO production was induced by the lipopolysaccharide
(LPS) covering the cell surface of M. loti (Nagata et al., 2009;
Murakami et al., 2011), but to date there is no evidence for an
induction of NO production by nodulation factors. The transient production of NO, which usually functions as a defenceinducible signal against bacterial pathogens, is related to the
expression of the LjHB1 gene, encoding the class 1 non-symbiotic hemoglobin (Hb1), which regulates the level of NO
(Nagata et al., 2008; Nagata et al., 2009; Murakami et al.,
2011). In contrast, inoculation of L. japonicus with the plant
pathogens Ralstonia solanacearum and Pseudomonas syringae
induces continuous NO production for at least 24 h (Nagata
et al., 2008). Treatments of L. japonicus roots with an NO
donor [S-nitroso-N-acetyl-D,L-penicillamine (SNAP)] and
NO scavenger (c-PTIO) resulted in the induction and repression, respectively, of LjHB1 (Shimoda et al., 2005; Nagata
et al., 2008). These observations led the authors to hypothesize that, at an early step of the symbiotic interaction,
the burst of NO specifically induces the expression of Hb1
which, in turn, downregulates the level of NO to lower the
plant defence response and allow the symbiont into the roots
(Murakami et al., 2011).
In the M. truncatula–S. meliloti interaction, both the scavenging of NO by c-PTIO, and the overexpression by the plant
partner of the bacterial flavohemoglobin hmp involved in NO
detoxification (Poole and Hughes, 2000), led to a delayed
nodulation phenotype (del Giudice et al., 2011), indicating
that NO is required for optimal establishment of symbiotic interaction and nodule development. In the same way,
Medicago plants with elevated NO levels generate more nodules than control plants (Pii et al., 2007). The reduction of
soybean nodule number by 70% following inoculated root
treatment with the NOS inhibitor L-NNA, and the reversion
of this reduction by addition of the NO donor diethylenetriamine NONOate (DETA-NO), led to similar conclusions
(Leach et al., 2010). However, Uchiumi’s team reported an
increase in the number of nodules formed on L. japonicus
transgenic hairy roots overexpressing either LjHb1 or Alnus
firma AfHb1 compared to those formed in control roots
(Shimoda et al., 2009). They concluded that NO was deleterious to nodule production. The apparent discrepancy with
the above-mentioned data may be explained by differences
in the symbiotic models, the timing of the observations in
the symbiotic process, and in the specific activity of either
flavo-hemoprotein (Hmp) or Hb. This underlines the need for
the setting up of an NO concentration range for successful
establishment of the symbiotic relationship. Furthermore, the
reduced expression of genes involved in nodule development
(MtCRE1, MtCCS52A) in NO-depleted roots reinforces the
hypothesis that NO could regulate the nodulation process
(del Giudice et al., 2011).
Transcriptomic studies have been performed to identify NO-responsive genes during the establishment of the
2880 | Hichri et al.
symbiotic interaction. A first study conducted on M. truncatula roots treated with two NO donors, sodium nitroprussiate (SNP) and S-nitroso-glutathione (GSNO), enabled the
identification of 999 putative NO-responsive genes (Ferrarini
et al., 2008). Of these genes, 290 were also regulated during
nodule development. Various genes involved in the developmental programme of the root hair during nodulation
[kinases, receptor-like kinases, and transcription factors
(TFs)], in carbon metabolism (sucrose transport, sucrose
synthase, or malate dehydrogenase), as well as in proteasome-dependent proteolysis were upregulated by NO. Genes
involved in the control of the cellular redox state, such as glutathione (GSH) and H2O2 metabolism, are also regulated by
NO, notably the two genes encoding the enzymes involved
in GSH synthesis, the γ-glutamylcysteine synthetase (γ-ECS)
and the glutathione synthetase (GSHS) (Innocenti et al.,
2007). More recently, a transcriptomic analysis using RNASeq technology was performed with M. truncatula-inoculated roots treated with c-PTIO to identify genes potentially
regulated by NO during nodule primordium development
(Boscari et al., 2013a). A comparison of NO-depleted and
non-depleted inoculated roots revealed differential patterns
of expression for 2030 genes. NO depletion prevented the
upregulation of many genes usually induced by inoculation with the microsymbiont. Many of these genes encode
TFs and proteins involved in nodule development, such as
cyclin-like proteins, peptidases, or ribosomal protein families, most of which are required for the dedifferentiation of
cortical cells and the induction of cell division during nodule
formation. The most striking finding of this study lies in the
reversal, upon c-PTIO treatment, of the downregulation of
defence genes normally triggered by inoculation with rhizobia (Fig. 2). The expression of a number of genes involved
in terpene, flavonoid, and phenylpropanoid pathways, and
genes encoding PR-proteins and cytochrome P450, were significantly affected by c-PTIO (Fig. 2). These observations
indicate that NO could be involved in the repression of plant
defence reactions, thereby favouring the establishment of the
beneficial plant–microbe interaction. This potential means of
NO action is similar to that proposed to occur in the mycorrhizal symbiosis (Espinosa et al., 2014), but differs markedly
from the signalling functions of NO in pathogenic interactions, in which NO cooperates with reactive oxygen species
(ROS) to induce hypersensitive cell death (Delledonne et al.,
1998).
NO in N2-fixing nodules
NO in N2-fixing nodules is provided by plant and
bacterial partners
NO accumulates in the N2-fixing zone of mature nodules of
M. truncatula and L. japonicus, and to a greater extent in
bacteroid-containing cells (Baudouin et al., 2006; Shimoda
et al., 2009). The concentration of NO in nodules has not
been determined precisely, but is estimated to be in the micromolar range (Meilhoc et al., 2010). In mature nodules, NO
originates from different sources, and its content is fine tuned
by the plant and bacterial partners (Horchani et al., 2011). The
application of (NG)-monomethyl-L-arginine (L-NMMA), a
competitive NOS inhibitor, on M. truncatula nodule slices led
to a drastic reduction of NO content (Baudouin et al., 2006).
Likewise, L. albus nodule extracts were able to produce NO
and L-citrulline when incubated with L-[14C] arginine, and
addition of L-NMMA had a significant inhibitory effect
on this production (Cueto et al., 1996), strengthening the
hypothesis of a NOS-like protein in nodules. In addition, the
potential involvement of PAOx in NO production has been
investigated in M. truncatula nodule slices, using the DAF2DA probe. The addition of spermine triggered an increase
in NO content, whereas that of gazatine (a PAOx inhibitor)
strongly reduced it, indicating that PAOx could be another
source of NO in mature nodules (Boscari, unpublished).
Additional sources of NO biosynthesis in nodules based
on a reductive pathway have been reported in plants, and
involve both NR and mitochondrial and bacterial ETCs
(Horchani et al., 2011). Incubation of M. truncatula nodules
with the NR inhibitor Tg significantly reduced NO production. Lack of MtNR1/2 expression in M. truncatula nodules
also affected the NO content, illustrating the participation of
these enzymes in NO production (Horchani et al., 2011). NR
inhibition by Tg can be alleviated in the presence of nitrite,
revealing that NR does not produce NO by itself. Under
hypoxic conditions, nitrite constitutes an alternative electron
acceptor of the ETC (Gupta and Kaiser, 2010). Hypoxia or
flooding conditions trigger NO accumulation in M. truncatula
or soybean nodules, but also in isolated bacteroids, together
with nitrite accumulation (Meakin et al., 2007; Sanchez et al.,
2010; Horchani et al., 2011), while they strongly induce NR
activity in soybean nodules (Sanchez et al., 2010). The use of
different inhibitors of the mitochondrial and bacteroid ETCs
revealed that both are involved in NO production (Horchani
et al., 2011). These studies indicate that, in M. truncatula
nodules, NO3– (nitrate) is reduced to NO2–, which is subsequently reduced to NO via the mitochondrial and bacteroid
ETCs (Horchani et al., 2011).
In the bacterial partner, the main pathway for NO production is by denitrification (Meakin et al., 2007; Sanchez
et al., 2010). The bacterial denitrification pathway includes
the napEDABC, nirKS, norCBQD, and nosRZDFYLX gene
clusters, encoding NR, nitrite reductase (NiR), NO reductase
(Nor), and nitrous oxide (N2O) reductase (Nos), respectively
(Bueno et al., 2012). Free living S. meliloti bacteria are able to
produce NO (Pii et al., 2007; Meilhoc et al., 2010), and NO
production measured on fresh nodule slices was not affected
in M. truncatula nodules developed from inoculation with the
S. meliloti nifH mutant strain, deficient in N2-fixation activity,
or with the nirK and norD strains, suggesting that N2-fixation
activity and bacterial denitrification are only minor sources
of NO in nodules (Baudouin et al., 2006). However, Horchani
et al. (2011) showed that in nodules resulting from infection
with bacterial napA or nirK strains, NO production of the
entire nodule was decreased by about 35% compared with
that of the wild-type strain. Comparable results emphasizing
NO production by the bacterial partner have been observed
Nitric oxide in N2-fixing symbiosis | 2881
Fig. 2. Comparison of transcriptional regulation of gene families in M. truncatula roots inoculated with S. meliloti: effect of c-PTIO treatment. (A)
Transcriptional regulation of gene families in inoculated roots (4 dpi) compared to non-inoculated roots. (B) Transcriptional regulation of gene families in
inoculated roots (4 dpi) treated for 8 h with 1 mM c-PTIO compared to inoculated roots (4 dpi). The classification of each gene in the families is based on
the protein function indicated in the Mt3.0 annotation. Nb, number; cdc, cell division control. Figure adapted from Boscari et al. (2013a) Plant Physiology
161, 425–439 (www.plantphysiol.org; Copyright American Society of Plant Biologists).
Fig. 3. Schematic representation of some effects of NO in N2fixing nodules. NO inhibits N2 fixation, and both carbon and nitrogen
metabolism, and activates GSH synthesis. Through the denitrification
pathway and the Hb-NO respiration cycle, NO favours the preservation
of an elevated energy state (ATP/ADP ratio). Dashed lines with + indicate
activation/induction/preservation effects of NO; dashed lines with –
indicate inhibition/repression effects of NO. Thick arrows indicate major
NO metabolic pathways. γ-EC, γ-glutamyl cysteine; γ-ECS, γ-glutamyl
cysteine synthetase; Gln, glutamine; ns-Hb, non-symbiotic hemoglobin;
Nif, nitrogenase; TCA, tricarboxylic acid.
in Glycine max–B. japonicum nodules (Sanchez et al., 2010).
Consequently, discrepancies regarding the importance of the
NO produced by rhizobia may be a result of the samples used
for NO detection (nodules slices vs entire nodules), as sample preparation can disturb microoxic conditions within the
nodule.
Effects of NO in developed nodules
The biological significance of NO in the symbiotic nodule has
been a matter of continual debate over recent years. NO was
first reported to be a potent inhibitor of nitrogenase activity
in vitro (Trinchant and Rigaud, 1982). It was also reported
to inhibit in vivo N2-fixing activity [measured through acetylene-reducing activity (ARA)] in soybean, L. japonicus, and
M. truncatula nodules (Trinchant and Rigaud, 1982; Shimoda
et al., 2009; Kato et al., 2010; Cam et al., 2012). Interestingly,
ARA is more substantial in L. japonicus nodules in the presence of 0.1 mM SNP than in the presence of lower (0.01 mM)
or higher (1 mM) concentrations of SNP, illustrating that
low but significant NO contents are necessary for N2 fixation (Kato et al., 2010). Soybean nodules exposed to flooding
showed increased NO contents, concomitant with a strong
reduction of bacterial nifH expression that could be partially restored by c-PTIO treatment, revealing that NO inhibits bacterial nitrogenase expression (Sanchez et al., 2010).
In S. meliloti, the flavohemoglobin hmp gene is induced by
NO. Hmp is essential for NO detoxification, and coordinates
the symbiosis event by modulation of NO levels in bacteria
and mature nodules (Meilhoc et al., 2010; Cam et al., 2012).
Compared to wild-type M. truncatula nodules, hmp-mutant
nodules displayed a 42%-enhanced NO content, and exhibited a drastically reduced ARA. As a result, plant growth was
reduced by 50%. In contrast, hmp++ nodules showed a 26%
reduction of NO content, and plants inoculated with hmp++
showed improved growth, indicating that excessive NO accumulation in mature nodules negatively affects N2-fixation efficiency and plant development (Cam et al., 2012).
Regulation of the nitrogenase activity by NO is believed
to occur through S-nitrosylation post-translational modifications, as suggested by computational prediction of
S-nitrosylation protein sites (Xue et al., 2010). Puppo et al.
(2013) reported that about 80 S-nitrosylated proteins have
been identified in mature nodules of M. truncatula. Many of
these proteins were related to nitrogen, carbon, and energy
metabolism in both plant and bacterial partners, emphasizing the crucial regulatory roles of NO in the modulation of
enzymatic activity (Fig. 3). Nitrogenase activity during the
N2-fixation process generates ammonium (NH4+), which at
high concentrations becomes toxic and compromises plant
growth (Li et al., 2014). Cytosolic glutamine synthetase (GS1)
plays a crucial role in nitrogen metabolism, as it assimilates
ammonium. In M. truncatula, GS1a and GS1b have been
identified, and MtGS1a is responsible for 90% of the total
2882 | Hichri et al.
nodule GS activity (Carvalho et al., 2000). In M. truncatula
nodules, MtGS1a can be inactivated by NO through tyrosine
nitration (Melo et al., 2011). This post-translational modification irreversibly abolishes MtGS1a enzymatic activity and
affects N2 fixation. In addition, glutamate (Glu), which is
necessary for GS activity, is also a precursor for the biosynthesis of the glutathione (GSH) that is both a major antioxidant compound and the precursor of GSNO. Consequently,
NO-mediated inactivation of the GS activity may promote
the redirection of the Glu pool for GSH and GSNO synthesis
(Fig. 3) (Melo et al., 2011). In addition, it has been reported
that GS nitration is enhanced in hmp-mutant nodules (Silva
and Carvalho, 2013). As hmp-mutant nodules exhibited premature senescence (Cam et al., 2012), it is tempting to associate NO-induced GS inhibition and nodule senescence. In
M. loti, GS1 deficiency induced premature nodule senescence
together with increased cysteine protease (CP) expression,
decreased Lb expression, and reduction of the N2-fixation
activity (Chungopast et al., 2014). Altogether these studies
reveal that NO seems to be required both for optimal symbiosis and for the regulation of nodule senescence.
Although average nodule lifespan is 10–12 weeks, their
N2-fixing capacity starts to decline 3–5 weeks after initiation
[reviewed in Puppo et al. (2005)]. Consequently, extending
nodule lifespan and the active N2-fixation period by fine tuning the senescence process may have beneficial effects on crop
yield. A typically visible sign of nodule senescence is its colour, changing from pink to green as a result of disruption of
Lb activity. In ageing soybean nodules, green Lb arises from
heme nitration, underlining the critical role of reactive nitrogen species in the senescence process (Meakin et al., 2007;
Navascues et al., 2012). Recent findings have identified NO
as a regulator of nodule aging. High NO contents induce premature senescence of M. truncatula nodules, while reduced
NO contents delay senescence, as observed by modulation of
nodule NO contents (Cam et al., 2012). Likewise, exogenous
application of an NO donor provoked premature senescence
of nodules (Cam et al., 2012). Developmental senescence in
M. truncatula nodules is initiated in a few scattered cells at the
centre of the fixation zone, before progressively extending to
the nodule periphery according to a conical front that follows
nodule growth (Perez Guerra et al., 2010); this makes it very
difficult to detect NO in the central cells and thus to ascertain
its putative role in the onset of senescence.
Characteristic key markers of the senescence process
include CPs, as they take part in the proteolytic activity and
thus resource remobilization. In M. truncatula, MtCP1 to
MtCP6 seem to play such a role, as revealed by their expression in senescing nodules (Van de Velde et al., 2006; Perez
Guerra et al., 2010), and MtCP6 has recently been further
characterized as a regulator of nodule senescence (Cam et al.,
2012; Pierre et al., 2014). Among additional identified proteases, MtVPE (vacuolar processing enzyme) is involved in
the regulation of nodule senescence in M. truncatula (Van
de Velde et al., 2006; Pierre et al., 2014). Although no direct
relationship between NO and proteolysis activity has been
reported so far in nodules, it has nevertheless been shown that
caspase 3-like activity is induced during abiotic stress-induced
cell death in Arabidopsis, and is actually activated by NO via
modulation of Mitogen-Activated Protein Kinase 6 activity
(Ye et al., 2013); similar results have been reported in barley
(Rodriguez-Serrano et al., 2012).
Because of its pleiotropic effects, NO detoxification represents a critical aspect of NO metabolism. Within the
symbiont, Lbs are in charge of maintaining a suitable microaerobic environment to allow O2 respiration and production
of energy without inactivation of the nitrogenase (Ott et al.,
2005). Despite their high affinity for O2, Lbs bind NO to
form LbNO (Mathieu et al., 1998). The LbNO complex is
present in large amounts in young nodules of soybean, and
decreases with nodule ageing or stressful environmental conditions, such as flooding (Meakin et al., 2007; Sanchez et al.,
2010). Lb has thus been proposed as an NO and peroxinitrite
scavenger, contributing to the protection of the nitrogenase
(Herold and Puppo, 2005; Sanchez et al., 2010). Functional
nodules are characterized by a microoxic environment, raising the question of energy supply within this organ. In plant
roots subjected to hypoxia, an alternative respiration cycle
known as ‘Hb-NO respiration’ occurs, where nitrite is used as
a final electron acceptor; this allows the retention of the cell’s
energy status (reviewed in Gupta and Igamberdiev, 2011).
The Hb-NO respiration cycle involves four steps (Fig. 4),
including (i) the reduction of NO3– by NR, (ii) the translocation of NO2– from the cytosol to the mitochondrial matrix,
(iii) the reduction of NO2– to NO via the mitochondrial ETC,
allowing ATP regeneration, and finally (iv) the passive diffusion of NO to the cytosol, where it is oxidized back to nitrate
by Hb (Gupta and Igamberdiev, 2011). Accumulating data
support the existence of such Hb-NO respiration in legume
nodules exposed to flooding, in which both mitochondrial
and bacterial ETCs are involved. Indeed, hypoxia or flooding conditions trigger NO accumulation in M. truncatula and
soybean nodules, and in isolated bacteroids (Meakin et al.,
2007; Sanchez et al., 2010; Horchani et al., 2011). In parallel,
a strong induction of LbNO complex formation is observed
in soybean root nodules (Meakin et al., 2007; Sanchez et al.,
2010), together with increased NR activity (Meakin et al.,
2007; Sanchez et al., 2010).
While Lbs are abundant in root nodules of leguminous
plants, other Hbs, such as Class 1 Hbs, have recently emerged
as being possibly involved in the symbiosis as well. L. japonicus LjHB1 is highly expressed in mature nodules (infected
and uninfected cells), and its expression correlates with NO
accumulation in nodules (Shimoda et al., 2005; Shimoda
et al., 2009). LjHB1 overexpression in L. japonicus increased
nodule number and consequently N2 fixation, associated with
reduced NO accumulation in the nodule (Shimoda et al.,
2009). Based on the pattern of Hbs expression during the
symbiotic process and under hypoxia, and on the fact that
Hbs can react with NO to form methemoglobin, it was suggested that Hb may be principally involved in NO scavenging
(Shimoda et al., 2009; Bustos-Sanmamed et al., 2011). NO
is also involved in the bacterial hypoxia response. In fact,
NO upregulates more than 100 genes in S. meliloti, including
genes of the denitrification pathway (Pii et al., 2007; Meilhoc
et al., 2010). Interestingly, 70% of the bacterial genes induced
Nitric oxide in N2-fixing symbiosis | 2883
Fig. 4. Schematic representation of the Hb-NO respiration and the bacteroid denitrification pathway in N2-fixing nodules. In the cytosol, NR reduces
NO3– to NO2–, which is transported to the mitochondria. NO2– is subsequently reduced into NO by the mitochondrial ETC. NO freely diffuses into the
cytosol where it is oxidized back into NO3– by either Lb or non-symbiotic Hb. This cycle allows ETC functioning and energy (ATP) regeneration in the plant
partner. Possible involvement of a plant NOS-like enzyme in NO production remains hypothetical. In the bacteroid, the denitrification route is at the start
of energy supply. NO3– is successively reduced into NO2–, NO, N2O, and N2, by nitrate reductase (Nap), nitrite reductase (Nir), NO reductase (Nor) and
nitrous oxide reductase (Nos). In the cytosol, the flavohemoglobin Hmp oxidizes NO into NO3–. The production of NO via a bacterial NOS-like enzyme
is also hypothetical. PEP, phosphoenolpyruvate; Arg, arginine; NiT, nitrite transporter; Nif, nitrogenase; IMS, mitochondrial intermembrane space; PBM,
peribacteroid membrane; PBS, peribacteroid space. The dotted lines indicate a possible movement of molecules between compartments.
by NO are also upregulated by hypoxia, and 50% of these
genes are dependent on the two-component system FixLJ,
regulating the O2-limitation response in S. meliloti (Meilhoc
et al., 2010).
Future prospects
NO in mycorrhizal and lichen symbioses
Increasing evidence shows that NO is produced in other symbiotic interactions, including mycorrhizal and lichen symbioses, and that it could play similar roles to that reported in the
N2-fixing symbiosis. In fact, NO was shown to be produced in
the roots of M. truncatula within minutes following treatment
with exudates of the mycorrhizal fungus Gigaspora margarita, and was suggested to be specifically related to mycorrhizal interaction (Calcagno et al., 2012). Similar observations
were made during the first steps of olive tree (Olea europea)–
Rhizophagus irregularis (Espinosa et al., 2014) and Trifolium
repens–Gigaspora mosseae (Zhang et al., 2013) symbiosis. As
mentioned for N2-fixing symbioses (Boscari et al., 2013b), NO
was suggested to be involved in the downregulation of defence
reactions to facilitate the establishment of the mycorrhizal
symbiosis between plant and fungus partners (Espinosa et al.,
2014). Although sources of NO have been poorly investigated
for the mycorrhizal symbiosis, NO was suggested to be produced by the coordinated activity of plasma membrane NR
and nitrite:NO reductase during the colonization of tobacco
roots by G. mosseae (Moche et al., 2010). This hypothesis
is supported by the observation that, in the M. truncatula–
G. margarita interaction, MtNR was upregulated during the
first hours following fungal inoculation, and that NO production is partially inhibited by Tg (Calcagno et al., 2012). NO
production was also investigated in lichens resulting from the
association of a fungus (mycobiont) and a photosynthetic
partner (photobiont) that may be an alga or cyanobacterium.
Weissman et al. (2005) showed that rehydration of the naturally desiccated lichen Ramalina lacera resulted in a burst of
ROS in both symbiotic partners, and of NO formation in the
hyphae of the fungal partner. NO-scavenging experiments
carried out in the lichen R. farinacea suggest that NO could
play a role in the photo-oxidative protection of the photobiont against peroxides generated during lichen rehydration
(Catala et al., 2010, 2013). Although the source of NO during
rehydration is presently unknown, effective communication
between mycobiont and photobiont, in which one partner
upregulates the antioxidant system of the other, has been
reported (Kranner et al., 2005). It was hypothesized that NO
could be produced in one partner, traverse the membrane, and
potentially activate a signal transduction cascade in the other
partner (Catala et al., 2013). Accumulating data show that in
both mycorrhizal and lichen symbioses, NO, together with
ROS, is part of global processes regulating the relationship
and the association between the two partners. Comparison
between pathogenic and symbiotic interactions will allow a
deciphering of the role of NO in the molecular processes leading to compatible versus incompatible relationships between
plants, fungi, and other microbial partners.
2884 | Hichri et al.
NO and ROS crosstalk
Crosstalk between ROS and NO appears to be another metabolic and signalling key for deciphering the regulation of
symbiosis. Combined ROS/NO regulation has already been
shown during plant–pathogen (Asai and Yoshioka, 2008;
Bellin et al., 2013), N2-fixing (Puppo et al., 2013) and mycorrhizal (Espinosa et al., 2014) interactions. In the N2-fixing
symbiosis, ROS and NO production have been detected
during the early phase of the interaction, as well as in the
nodulation process, and both H2O2 and NO have been suggested to be involved in the senescence of the nodule (Puppo
et al., 2013). It may be underlined that many of the proteins
identified as being S-nitrosylated in the symbiotic interaction
have also been reported to be S-sulfenylated (Puppo et al.,
2013), suggesting that the same protein may be differentially
regulated depending on the redox state. The regulation of
nodule NADPH oxidase activity by NO (Yun et al., 2011;
Marino et al., 2012) is an important link between NO and
O2–. In terms of reactive species, peroxynitrite is a reactive
nitrogen species formed when NO reacts with O2–. To date,
peroxynitrite is emerging as a potential signalling molecule in
the induction of defence response against pathogens, and this
could be mediated by the selective nitration of Tyr residues in
a small number of proteins (Vandelle and Delledonne, 2011).
As both NO and O2– are produced in symbiotic nodules, it
is conceivable that peroxynitrite is formed in these organs.
Lb was shown to scavenge peroxynitrite, thus precluding any
damaging effect of this species in the nodules (Herold and
Puppo, 2005). The demonstration that GS1a is nitrated in
nodules, whereas GS2a is subjected to S-nitrosylation, provides a direct link between NO/O2– signalling and nitrogen
metabolism in root nodules (Melo et al., 2011). Peroxynitrite
signalling appears to be an interesting field of investigation
for understanding redox regulation of N2 fixation.
NO and hormone crosstalk
The crosstalk between NO and various phytohormones during the different steps of nodule establishment and functioning is emerging as another promising field of investigation.
A first link between NO and auxin (IAA) has been shown
during initial steps of the symbiosis. An S. meliloti strain
overexpressing a bacterial IAA biosynthesis gene promotes
the formation of M. truncatula nodules and root growth, and
NO accumulates 2-fold more in IAA-overproducing nodules
than in control nodules (Pii et al., 2007). Since no difference in
NO content has been detected between wild-type and IAA–
rhizobia strains, the increase of nodule NO is likely to be due
to the plant partner (Pii et al., 2007). More recently, NO was
found to interfere with the cytokinin signalling pathway in the
regulation of M. truncatula nodule development (del Giudice
et al., 2011). Regulatory effects of NO on cytokinin signalling may occur via inhibition of the phosphotransfer protein
activity through S-nitrosylation, as described for Arabidopsis
AHP1 (Feng et al., 2013). Additional reciprocal regulatory
events probably exist: Arabidopsis continuous NO-unstressed1
mutants, which are less responsive to NO, display elevated
cytokinin contents; zeatin treatment rescues the nitric oxide
overexpression 1 phenotype; and a physical interaction
between zeatin and peroxynitrite has been reported, suggesting that cytokinin can act as an NO repressor in Arabidopsis
(Liu et al., 2013). Likewise, the L. japonicus enhanced nitrogen fixation 1 (enf1) mutant showed an enhanced number and
weight of root nodules, but also N2 fixation and plant growth,
as a result of low endogenous ABA contents (Tominaga et al.,
2009). NO production was decreased in enf1 nodules, which
may increase the N2 fixation activity (Tominaga et al., 2009).
Finally, NO was reported to regulate both ethylene and jasmonic acid biosynthesis pathways in plants through enzyme
S-nitrosylation and regulation of biosynthetic gene expression (reviewed in Mur et al., 2013). Considering that NO was
identified as a key regulator of nodule senescence (Cam et al.,
2012), and ethylene and jasmonic acid biosynthesis genes are
specifically induced in senescent nodules (Van de Velde et al.,
2006), it is tempting to hypothesize that NO could be part
of the signalling pathway regulating ethylene and jasmonic
acid synthesis at the onset of nodule senescence. Deep and
extended investigations are still needed, but the intriguing
relationships between NO and hormones are progressively
becoming better known.
NO perception
As already mentioned, fine tuning of NO responses is highly
dependent on the NO concentration, which raises the question of its perception. Until recently, it was thought unlikely
that NO could interact with a single defined receptor (BessonBard et al., 2008). The recent identification of a general
mechanism of NO sensing in plants constituted a milestone
in plant physiology (Gibbs et al., 2014). Gibbs and coworkers identified the group VII of Ethylene Response Factors
(ERFs-VII) as central hubs for the perception of the NO signal in plants. The targeted proteolysis of ERFs-VII by the
N-end rule pathway was first proposed as an indirect mechanism for O2 sensing (Gibbs et al., 2011; Licausi et al., 2011),
but it has now been demonstrated that both O2 and NO are
required to destabilize these proteins and that a reduction in
the availability of either gas is sufficient (Gibbs et al., 2014).
The exact sequence of NO- and O2-mediated Cys modifications that lead to substrate destabilization via the N-end
rule pathway is unknown, but by comparison with a mammalian model (Hu et al., 2005), authors raise the hypothesis that the N-terminal Cys is first S-nitrosylated by NO
and then further oxidized by O2 to produce Cys sulfinic and
Cys sulfonic acids that can then be arginylated (Gibbs et al.,
2014). A recent study in plants showed that plant cysteine
oxidases, which share homology with thiol oxidases found in
animal cysteine dioxygenases, specifically oxidize N-terminal
Cys residues using O2 as a cosubstrate (Weits et al., 2014).
Furthermore, Gibbs and coworkers showed that alteration in
NO levels, brought about by changes in NR activity, is sufficient to modulate ERFs-VII stability, suggesting that NR
could be the main source of NO involved in signalling (Gibbs
et al., 2014). The authors suggested that N-end rule control
of ERFs-VII stability may therefore provide a way for plants
Nitric oxide in N2-fixing symbiosis | 2885
Fig. 5. Summary of sources of NO and its main functions in developing and mature N2-fixing nodules. Putative NO sources are indicated with question
marks. NirK, nitrite reductase K.
to link metabolism to gene expression and it is possible that
this mechanism may play a role in plant responses to nitrate
availability. To date, no work was undertaken on ERFs-VII
in N2-fixing symbioses. In the M. truncatula genome (version Mt4.0: http://www.jcvi.org/medicago/), four genes that
belong to the ERFs-VII group have been identified (Boscari
et al., personal communication). Deduced proteins harbour
the conserved N-terminal amino acid motif shown to target
their fate under different O2 levels in Arabidopsis. Analysis
of the RNA-Seq database (Boscari et al., 2013a) showed
that the four genes are expressed in both roots and nodules.
Interestingly, one of them is 5-fold more expressed in nodules
than in roots, suggesting that it could play a particular role in
microoxic nodules (Boscari et al., 2013b). These preliminary
data strongly suggest that ERFs-VII could fulfil the same
functions in legumes as their Arabidopsis counterparts, and
appear to be particularly interesting candidates for deciphering O2 perception and NO signalling in N2-fixing symbioses.
Concluding remarks
NR and NOS-like activities were shown to be NO sources
during the early steps of the establishment of symbioses. In
contrast, the bacterial denitrification pathway and the plant
NR/mitochondrial ETC system (i.e. reductive pathways
of NO production) appear to be the main source of NO in
the microoxic environment of the N2-fixing mature nodule
(Fig. 5). The presence of NOS-like or PAOx activities, i.e. oxidative NO-production pathways, has been shown in mature
nodule extracts or slices, but their physiological meaning
remains to be determinated in vivo. Considered as a whole,
the literature shows that NO acts as a multifaceted regulator
involved in many mechanisms of the symbiotic process: symbiont recognition; modulation of plant defence reactions; cell
division and organogenesis during symbiosis establishment;
energy, nitrogen, and carbon metabolism in mature nodules;
and in the onset of senescence. Increasing evidence show that
these mechanisms are common to various symbiotic interactions, and that NO interacts with other major regulators of
plant growth and development such as hormones and ROS.
Deciphering these interactions and the mechanisms of NO
perception and signalling appears to be the challenging issue
for the next decade.
Funding
This work was supported by the Institut National de la
Recherche Agronomique, the Centre National de la Recherche
Scientifique, the University of Nice–Sophia Antipolis, and
the French Government (National Research Agency, ANR)
through the LABEX SIGNALIFE programme (reference #
ANR-11-LABX-0028-01). IH was supported by a post-doctoral fellowship from the Institut National de la Recherche
Agronomique.
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