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, France2 CNRS, Institut Sophia Agrobiotech (ISA), UMR 7254, BP 167, 06903, Sophia Antipolis cedex, France3 Université Nice Sophia Antipolis, Institut Sophia Agrobiotech (ISA), BP 167, 06903, Sophia Antipolis cedex, France

* To whom correspondence should be addressed. E-mail: renaud.brouquisse@sophia.inra.fr

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 micro-aerophilic 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 bacte-rial lipochito-oligosaccharides, termed ‘nodulation factors’,

bacteria penetrate root hairs via a specific structure, the infec-tion 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

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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, flavo-hemoprotein; 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 reduc-tase; PAOx, polyamine oxidase; PEP, phosphoenolpyruvate; SNP, sodium nitroprussiate; Tg, tungstate; wpi, weeks post-inoculation.

Journal of Experimental Botany, Vol. 66, No. 10 pp. 2877–2887, 2015doi:10.1093/jxb/erv051 Advance Access publication 1 March 2015

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 bacte-roids; 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 senes-cence (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 unin-fected 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 condi-tions prevailing in the nodules (Appleby, 1992). Thus, nodule development occurs in changing conditions, shifting from a normoxic environment during the establishment of the sym-biosis to a microoxic one in functioning nodules.

Nitric oxide (NO) is a free radical, reactive gaseous mol-ecule with a broad spectrum of regulatory functions in plant growth and development (Besson-Bard et  al., 2008), and responses to biotic (pathogenic and symbiotic interac-tions) 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-perme-able 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 sym-biotic 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 leghe-moglobin (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 nod-ules 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 pro-duced throughout the whole symbiotic process (Fig. 1), and the question was raised as to the origin of NO and its physi-ological roles in the different time and space of the symbiotic interaction.

Both the plant and the bacterial partners should be con-sidered as potential sources of NO. In plants, beside a non-enzymatic 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 membrane-bound nitrite:NO reductase, xanthine oxido-reductase, or the mitochondrial electron-transport chain (ETC), par-ticularly 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.

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polyamine oxidase (PAOx)], occurs under normoxic condi-tions (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 envi-ronment 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 sym-biotic 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 symbio-sis, from the early steps of the interaction to the senescence of the nodule. It goes on to present several particularly promis-ing 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 symbi-osis 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-L-arginine (L-NNA) negatively affects the interaction of soy-bean 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 charac-terized 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 symbi-otic 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 interac-tion 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-tetramethyl-imidazoline-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 tran-sient production of NO, which usually functions as a defence-inducible signal against bacterial pathogens, is related to the expression of the LjHB1 gene, encoding the class 1 non-sym-biotic 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 repres-sion, respectively, of LjHB1 (Shimoda et  al., 2005; Nagata et al., 2008). These observations led the authors to hypoth-esize 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 scav-enging 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 symbi-otic interaction and nodule development. In the same way, Medicago plants with elevated NO levels generate more nod-ules 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 diethylene-triamine 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 delete-rious 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 iden-tify NO-responsive genes during the establishment of the

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symbiotic interaction. A first study conducted on M. trunca-tula roots treated with two NO donors, sodium nitroprus-siate (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 devel-opmental 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 proteas-ome-dependent proteolysis were upregulated by NO. Genes involved in the control of the cellular redox state, such as glu-tathione (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 RNA-Seq technology was performed with M.  truncatula-inocu-lated 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 inocula-tion with the microsymbiont. Many of these genes encode TFs and proteins involved in nodule development, such as cyclin-like proteins, peptidases, or ribosomal protein fami-lies, 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 rhizo-bia (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 sig-nificantly 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 mycor-rhizal symbiosis (Espinosa et al., 2014), but differs markedly from the signalling functions of NO in pathogenic interac-tions, 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 micro-molar 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 DAF-2DA 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 produc-tion. 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 subse-

quently reduced to NO via the mitochondrial and bacteroid ETCs (Horchani et al., 2011).

In the bacterial partner, the main pathway for NO pro-duction 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

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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 sam-ple 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 acety-lene-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 pres-ence 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 fixa-tion (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 par-tially restored by c-PTIO treatment, revealing that NO inhib-its 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 exhib-ited 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 accu-mulation in mature nodules negatively affects N2-fixation effi-ciency and plant development (Cam et al., 2012).

Regulation of the nitrogenase activity by NO is believed to occur through S-nitrosylation post-translational modi-fications, 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, emphasiz-ing 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

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 N2-fixing 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.

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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 modifi-cation 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 biosyn-thesis of the glutathione (GSH) that is both a major antioxi-dant 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 pre-mature senescence (Cam et al., 2012), it is tempting to asso-ciate 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 symbio-sis 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 tun-ing the senescence process may have beneficial effects on crop yield. A typically visible sign of nodule senescence is its col-our, 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 nitro-gen 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 pre-mature 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 expres-sion 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 pro-teases, 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 rep-resents a critical aspect of NO metabolism. Within the symbiont, Lbs are in charge of maintaining a suitable micro-aerobic 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 con-ditions, 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, rais-ing 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 transloca-tion 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 diffu-sion 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 flood-ing 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. japoni-cus 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 sug-gested 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

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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 sym-biotic interactions, including mycorrhizal and lichen symbi-oses, 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 marga-rita, and was suggested to be specifically related to mycorrhi-zal 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 pro-duced 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 produc-tion 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 natu-rally 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 photo-biont 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 lead-ing to compatible versus incompatible relationships between plants, fungi, and other microbial partners.

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.

Nitric oxide in N2-fixing symbiosis | 2883

NO and ROS crosstalk

Crosstalk between ROS and NO appears to be another meta-bolic 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 myc-orrhizal (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 sug-gested 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, pro-vides 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 dur-ing the different steps of nodule establishment and function-ing 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 signal-ling 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, suggest-ing that cytokinin can act as an NO repressor in Arabidopsis (Liu et al., 2013). Likewise, the L. japonicus enhanced nitro-gen 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 jas-monic acid biosynthesis pathways in plants through enzyme S-nitrosylation and regulation of biosynthetic gene expres-sion (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 ques-tion of its perception. Until recently, it was thought unlikely that NO could interact with a single defined receptor (Besson-Bard 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 cowork-ers identified the group VII of Ethylene Response Factors (ERFs-VII) as central hubs for the perception of the NO sig-nal in plants. The targeted proteolysis of ERFs-VII by the N-end rule pathway was first proposed as an indirect mecha-nism 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 modifi-cations that lead to substrate destabilization via the N-end rule pathway is unknown, but by comparison with a mam-malian model (Hu et  al., 2005), authors raise the hypoth-esis 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 suf-ficient 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

2884 | Hichri et al.

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 (ver-sion 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 decipher-ing 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. oxi-dative 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: sym-biont 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 interac-tions, 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-doc-toral fellowship from the Institut National de la Recherche Agronomique.

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