Redox Changes during the Legume–Rhizobium Symbiosis

Molecular Plant Volume 2 Number 3 Pages 370–377 May 2009 REVIEW ARTICLE 

Redox Changes during the Legume–Rhizobium 

Symbiosis 

Christine Chang, Isabelle Damiani, Alain Puppo and Pierre Frendo 1 

UMR INRA–Université de Nice–Sophia Antipolis–CNRS ‘Interactions Biotiques et Santé Végétale’, 400 Route des Chappes, BP167, 06903 Sophia Antipolis Cedex, 

France 

ABSTRACT Reactive Oxygen Species (ROS) are continuously produced as a result of aerobic metabolism or in response to 

biotic and abiotic stresses. ROS are not only toxic by-products of aerobic metabolism, but are also signaling molecules 

involved in plant growth and environmental adaptation. Antioxidants can protect the cell from oxidative damage by scavenging 

the ROS. Thus, they play an important role in optimizing cell function by regulating cellular redox state and modifying 

gene expression. This article aims to review recent studies highlighting the role of redox signals in establishing and 

maintaining symbiosis between rhizobia and legumes. 

Key words: Oxidative and photo-oxidative stress; cell differentiation/specialization; gene regulation; symbiosis; legume. 

NITROGEN FIXING SYMBIOSIS 

Nitrogen, a component of many bio-molecules, is essential for 

growth and development of all organisms. Most nitrogen 

exists in the atmosphere, and utilization of this source is important 

in avoiding nitrogen starvation. However, the ability to fix 

atmospheric dinitrogen (N2) via the nitrogenase enzyme complex 

is restricted to certain types of diazotrophic archeobacteria 

and eubacteria. Rhizobia, a type of gram-negative soil 

bacteria, are able to reduce atmospheric nitrogen through 

a symbiotic interaction with plants. They are located within 

the Rhizobiaceae phylogenetic family (a-proteobacteria) and 

have the unique ability to infect and establish symbiosis with 

leguminous plants. This symbiosis reduces the need for nitrogen 

fertilizers for agriculturally important plants such as soybean 

and alfalfa. 

The symbiotic interaction is initiated when the bacterium 

infects the root hair, and the host forms root nodules where 

nitrogen fixation will occur (Dénarié and Cullimore, 1993). 

In order for this to happen, the host must first identify rhizobia 

as beneficial partners (Figure 1A). Plants secrete (iso)flavonoids 

that are recognized by the compatible bacteria. They bind and 

activate bacterial NodD proteins, which are members of the 

LysR family of transcriptional regulators, resulting in the induction 

of the nodulation genes (nod genes) (Peck et al., 2006). 

Nod genes encode proteins that synthesize and export specific 

lipochito-oligosaccharides called Nod factors (NFs). NFs activate 

the root infection process and initiate cell division in 

the root cortex. NFs are not the only bacterial signals necessary 

for symbiosis: surface lipopolysaccharides (LPS) also contribute 

at various stages of symbiotic development (Oldroyd and 

Downie, 2008). 

Host recognition of the NFs leads to increased calcium levels 

in the root hairs, followed by strong calcium spiking, and lastly 

alteration of the root hair cytoskeleton (Sieberer et al., 2005). 

Plant root hairs curl and trap rhizobial bacteria and induction 

of root cortical cell division establishes a meristem and nodule 

primordium (Figure 1B). The bacteria within this colonized 

curled root hair induce the formation of infection threads that 

are ingrowths of the root-hair cell membrane filled with bacteria 

(Figure 1C). In the inner plant cortex, bacteria exit the infection 

thread and enter nodule cells by endocytosis. Each 

bacterial cell is endocytosed in a membrane-bound compartment 

(Brewin, 2004). This organelle-like structure, including 

the plant-derived membrane and the bacteroid (the differentiated 

bacterium), is called the symbiosome. An infected plant 

cell may be packed with thousands of symbiosomes. 

This bacteroid is provided with a low oxygen environment 

that allows expression of enzymes of the nitrogenase complex 

and thus nitrogen fixation (Fischer, 1994). This microaerobic environment 

within the nodule is adjusted by a plant-produced 

1 

To whom correspondence should be addressed. E-mail frendo@unice.fr, 

fax +33-492386587, tel. +33-492386638. 

ª The Author 2008. Published by the Molecular Plant Shanghai Editorial 

Office in association with Oxford University Press on behalf of CSPP and 

IPPE, SIBS, CAS. 

doi: 10.1093/mp/ssn090, Advance Access publication 26 December 2008 

Received 18 September 2008; accepted 18 November 2008 

Downloaded from 

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oxygen-binding protein, the leghaemoglobin (Downie, 2005; 

Ott et al., 2005). Bacteroid differentiation is concomitant with 

the down-regulation of most metabolic processes and an increase 

in the gene products involved in nitrogen fixation 

and respiration. Respiratory activity provides nitrogenase with 

16 molecules of ATP and eight electrons that are estimated to 

be required to reduce one molecule of N2 to two molecules of 

NH4 + (Jones et al., 2007). Ammonium is secreted by the bacteroid 

and assimilated by plant cells through primarily glutamine 

and asparagine synthetases. Carbon is supplied by the plant 

in the form of a dicarboxylic acid such as malate and this provides 

metabolites and energy for bacteroid differentiation and 

nitrogen fixation (Jones et al., 2007). 

MODULATION OF REACTIVE OXYGEN 

SPECIES DURING NITROGEN FIXING 

SYMBIOSIS 

Reactive Oxygen Species (ROS) are constantly produced by 

plants as a consequence of aerobic metabolism during their 

development or in response to abiotic and biotic stresses. 

ROS includes singlet oxygen ( 1 O2), superoxide anion (O2 .– ), hydrogen 

peroxide (H2O2), and hydroxyl radical (OH . ) species. 

These molecules are highly toxic, since they are able to modify 

all the primary constituents of the cell such as lipids, DNA, carbohydrates, 

and proteins (Moller et al., 2007). Their toxicity 

leads to senescence and cell death (Overmyer et al., 2003; Rivero 

Chang et al. d Redox Changes during the Legume–Rhizobium Symbiosis | 371 

Figure 1. The Different Steps of Nitrogen Fixing 

Symbiosis. 

(A) Recognition of the partners. 

(B) Root hair curling. 

(C) Infection thread growth. 

(D) M. truncatula nodule with meristematic 

(I), infection (II), fixing (III), and senescent 

(IV) zones. NFs, Nod factors. 

et al., 2007). ROS are also involved in the regulation of plant 

metabolism as secondary messengers in many pathways associated 

with plant development and environmental stress 

responses (Apel and Hirt, 2004; Carol and Dolan, 2006; Gechev 

et al., 2006). Such ROS production may be controlled and deliberate, 

enabling plants to better adapt to the environment 

(Pitzschke et al., 2006). 

Accumulation of ROS has been observed in Medicago sativa 

(alfalfa) during the interaction between rhizobia and legumes 

(Santos et al., 2001). The ROS accumulation, detected 12 h 

post treatment with NFs, is linked to the NFs signaling transduction 

pathway as S. meliloti mutants that produce altered 

NFs or a non-nodulating Medicago truncatula mutant, 

dmi1-1, are impaired in the ability to elicit ROS production 

(Ramu et al., 2002). Recently, fast and transient ROS changes 

were observed in root hair cells, minutes after treatment with 

NFs in Phaseolus vulgaris (Cardenas et al., 2008). Inhibition of 

this ROS production prevents root hair curling and formation 

of infection threads (Peleg-Grossman et al., 2007). Thus, the 

production of ROS may not be a plant defense response to 

the microbe, but rather a process that is needed for the development 

of a proper interaction. The importance of ROS 

production has been confirmed in a S. meliloti strain overexpressing 

a catalase. This bacterium, acting as an H2O2 sink, provoked 

a delayed nodulation and enlargement of infection 

threads (Jamet et al., 2007). Hydrogen peroxide production 

may be involved in cell wall formation and in regulation of 



372 | Chang et al. d Redox Changes during the Legume–Rhizobium Symbiosis 

the infection thread rigidity that is needed for its progression 

through the plant. The induction of genes encoding rhizobiuminduced 

peroxidase such as Rip1 (Cook et al., 1995) or Srprx1 

(Den Herder et al., 2007) is consistent with this hypothesis. 

In the semi-aquatic legume Sesbania rostrata, invasion may 

happen through fissures in the lateral root base by entry to the 

cortex via intercellular cracks (Goormachtig et al., 2004). In this 

model, the formation of infection pockets also depends on NFs 

and was shown to be associated with localized cell death and 

the production of large amounts of H2O2 (D’Haeze et al., 

2003). Moreover, pharmacological experiments showed that 

ROS were required for nodule initiation (D’Haeze et al., 2003). 

In contrast with these results describing the production of 

ROS during nodule development, NFs have been shown to inhibit 

the ROS efflux in M. truncatula during the very early steps 

of the interaction, 1 h post inoculation (Shaw and Long, 

2003). This down-regulation of early ROS accumulation is 

not observed in plants deficient for genes encoding proteins 

involved in the symbiotic interaction such as a protein kinase 

(CDPK1) or a putative NFs receptor NFP (Ivashuta et al., 2005), 

suggesting that it is linked to the NFs signaling transduction 

pathway. This reduction in ROS efflux was correlated with 

a transient decrease of transcript accumulation of two NADPH 

oxidase homologs, MtRBOH2 and MtRBOH3, 1 h after NFs 

treatment (Lohar et al., 2007). This transient down-regulation 

of both MtRBOH is not observed in the mutant nfp showing 

that the early stage of NFs perception is required for the regulation 

of MtRBOH2 and MtRBOH3. These results suggest that 

NFs suppress the very early activity of the ROS-generating system 

during the interaction and, thus, may allow a compatible 

interaction between the plant and the bacteria. Moreover, a S. 

meliloti strain deficient in the production of NFs and in the infection 

process induces H2O2 accumulation during the first 

hours post inoculation as compared with the wild-type S. meliloti 

(Bueno et al., 2001). Furthermore, treatment of a M. sativa 

suspension culture with yeast elicitors and S. meliloti LPS is unable 

to induce the ROS formation observed when the cells are 

treated with yeast elicitors alone, showing that LPS might 

modulate ROS production during the interaction (Albus 

et al., 2001). Finally, another S. meliloti mutant strain, which 

can not produce exopolysaccharides, is defective in invading 

the plant and triggers the induction of a larger number of 

genes belonging to the plant defense category in M. truncatula 

(Jones et al., 2008). Taken together, these results show that 

the absence of NFs or exopolysaccharides impairs the infection 

process, perhaps via a plant defense response that may be 

linked to ROS production. In conclusion, the differential 

ROS accumulation observed at different time points of the 

symbiotic interaction suggests that the ROS level may be involved 

in different processes at multiple spatiotemporal steps 

during nodule formation. 

The production of H2O2 during symbiosis was detected in 

infection threads, in the thread walls of both the infection 

and fixing zones of M. sativa and Pisum sativum nodules (Santos 

et al., 2001; Rubio et al., 2004). Hydrogen peroxide is also 

detected in the cell walls and intercellular spaces of the cortex. 

This production of H2O2 may be involved in the oxidative crosslinking 

needed for strengthening of the infection threads and 

cell wall formation. In contrast, H2O2 was not detected in the 

nitrogen-fixing zone of the nodules (Santos et al., 2001; Rubio 

et al., 2004). This lack of detection may be attributed to the 

strong antioxidant defense (see antioxidant defense in the 

next section) or to its fast reaction with biological compounds 

present in the nodule. Leghaemoglobin is a candidate for oxidative 

damage. 

Leghaemoglobin is present at a very high concentration in 

nodules where it facilitates oxygen transport to the bacteroids. 

Exposure of young or mature nodules to oxidative stress results 

in the formation of leghaemoglobin radicals that are similar to 

those observed during natural senescence (Mathieu et al., 

1998). Moreover, when linked to O2, this hemoprotein undergoes 

facile autoxidation to form ROS such as superoxide radicals 

or H2O2 (Puppo et al., 1981). This ROS formation is 

important, as plants deficient in the formation of leghemoglobin 

contain a significantly lower level of H2O2 (Gunther et al., 

2007). Multiple results suggest that redox balance is involved 

in the regulation of nodule metabolism. The diminution of nitrogen 

fixation under abiotic stress is correlated with a modification 

of the redox balance and a strong decline in the 

antioxidant defense (Escurado et al., 1996; Gogorcena et al., 

1997; Jebara et al., 2005). Moreover, this decrease in nitrogen 

fixation is associated with a carbon flux shortage (Matamoros 

et al., 1999a; Galvez et al., 2005). Sucrose synthase, which plays 

a major role in carbon metabolism regulation and is crucial for 

an adequate nitrogen fixation (Baier et al., 2007), has been 

shown to be regulated by redox state in nodules (Marino 

et al., 2008). Interestingly, modification of nodule metabolism 

under senescence could be partially mimicked by a direct oxidative 

stress on mature nodules (Mathieu et al., 1998; Marino 

et al., 2006). 

During nodule senescence, ROS have been detected around 

senescing symbiosomes, suggesting their involvement in this 

process (Alesandrini et al., 2003; Rubio et al., 2004). At the molecular 

level, a transcriptomic analysis performed with M. truncatula 

nodules showed that the transcriptomes of nodule and 

leaf senescence have a high degree of overlap, arguing for the 

recruitment of similar pathways (Van de Velde et al., 2006). 

Thus, a combination of multiple factors including ROS may 

be involved in the control of the events leading to the rupture 

of the symbiotic interaction (Puppo et al., 2005). 

INVOLVEMENT OF ANTIOXIDANT 

DEFENSE IN NITROGEN FIXING 

SYMBIOSIS 

Plant cells contain an impressive antioxidant defense with enzymatic 

(mainly water soluble) and non-enzymatic (water and 

lipid soluble) ROS scavenging systems. All types of antioxidants 

described in plants have also been detected in nodules. This 

includes: (1) enzymatic systems directly involved in ROS 



scavenging such as superoxide dismutase (SOD), catalase 

(KAT), and peroxidase (Prx); (2) the non-enzymatic antioxidant 

molecules such as ascorbate (ASC), glutathione (GSH), and 

a-tocopherol; (3) the ascorbate–glutathione pathway, which 

allows the reduction of the two antioxidant molecules by 

NAD(P)H; and (4) the enzymes involved in the disulfide reduction, 

thioredoxin (Trx) and glutaredoxin (Grx). The bacterial 

partner that differentiates to bacteroid to reduce atmospheric 

nitrogen also contains a large range of antioxidant systems including 

enzymatic and non-enzymatic defense. This section 

will focus on the involvement of the antioxidant systems in 

the following facets of nitrogen-fixing symbiosis: nodule formation, 

nitrogen fixing efficiency, and alteration of this efficiency 

under stress conditions. 

Antioxidant systems are known to be involved in the regulation 

of developmental processes because mutants in various 

antioxidant systems showed growth defect and organ malformations 

(for reviews, see Beveridge et al., 2007; Covarrubias 

et al., 2008). In this framework, the redox transduction pathway 

has been shown to play an important role in nodule development. 

Sinorhizobium meliloti genome-wide transcriptome 

experiments showed that sodC specifically is induced during 

Medicago infection (Ampe et al., 2003). Bacterial mutants deficient 

in catalase activity are impaired in their symbiotic efficiency 

(Sigaud et al., 1999; Jamet et al., 2003). S. meliloti contains three 

catalase genes (katA, katB, andkatC), which are differentially 

expressed during the various physiological states of the bacteria. 

Whereas the symbiotic efficiency of single mutant strains was 

not affected, the double mutant katA katC nodulated poorly, 

and the double mutant katB katC displayed abnormal infection 

with a failure in bacteroid differentiation. Nevertheless, recent 

results obtained during the symbiotic interaction between Medicago 

sativa and an overexpressing S. meliloti RmkatB ++ mutant 

indicate that H2O2 is required for optimal progression of infection 

threads through the root hairs and plant cell layers (Jamet 

et al., 2007). 

Antioxidant molecules have also been shown to be involved 

in symbiosis efficiency. In M. truncatula, inhibition of GSH and 

homoglutathione (hGSH) synthesis (a legume-specific GSH homolog) 

by pharmacological or genetic approaches inhibited 

root nodule formation (Frendo et al., 2005). A strong reduction 

in the number of nascent nodules and in the expression 

of the early nodulin genes, involved in nodule formation, was 

observed in GSH and hGSH-depleted plants. In parallel, the importance 

of GSH during symbiosis of S. meliloti and Rhizobium 

tropici has also been demonstrated (Harrison et al., 2005; 

Muglia et al., 2008). The S. meliloti and R. tropici strains deficient 

in gshB, the gene encoding the enzyme catalyzing the 

second step of GSH synthesis, were affected in their symbiotic 

efficiencies with a delayed nodulation coupled with a reduction 

in the nitrogen-fixation capacity. This phenotype was linked to 

abnormal nodule development with early nodule senescence. 

There are several indications that Trxs also play an important 

role in the establishment of the nitrogen-fixing symbiotic process. 

Recently, a detailed analysis of Trxs in M. truncatula estab- 


lished the existence of two isoforms that do not belong to any 

of the previously described types (Alkhalfioui et al., 2008). Interestingly, 

these novel isoforms seem to be specifically dedicated 

to the symbiotic interaction according to their pattern of expression. 

In Glycine max, a Trx was found to be involved in nodulation, 

as plants deficient in this isoform have a reduced number 

of nodules (Lee et al., 2005). The bacterial partner, a Bradyrhizobium 

japonicum mutant deficient in tlpA (a gene encoding 

protein having a Trx domain), was notably defective in the development 

of a nitrogen-fixing endosymbiosis and exhibited 

a strong decrease in oxidase activity compared to the wild-type 

(Loferer et al., 1993). All these data emphasize the role of the 

antioxidant defense and redox state regulation in the establishment 

of an efficient symbiosis between legumes and rhizobia. 

In the functional nodule, numerous processes may contribute 

to a high production of ROS. The highly reducing environment 

needed for nitrogen fixation may lead to ROS formation, 

as many electron donors, including ferredoxin, uricase, and hydrogenase, 

are susceptible to auto-oxidation, resulting in superoxide 

formation (Dalton et al., 1991). Oxyleghemoglobin, 

which is present at a high concentration in nodules, can also 

produce superoxide (Puppo et al., 1991). In addition, the high 

level of transition metals in the nodule may lead to the generation 

of ROS (Becana and Klucas, 1992). In this context, 

a strong antioxidant defense may be crucial in order to allow 

efficient nodule metabolism. As mentioned previously, H2O2 is 

not detected in the nitrogen-fixing zone of nodules. It has 

been proposed that antioxidant defense participates in the 

limitation of the entry of O 2 into the nodule interior (Dalton 

et al., 1998). Expression of ascorbate peroxidase (APX) is highly 

induced in soybean nodules in which APX represents about 1% 

of the protein (Dalton et al., 1987). The preferential localization 

of the APX is the endodermis of indeterminate nodules in 

alfalfa, pea, and clover and the exterior edge of the parenchyma 

of determinate nodules in bean and soybean. These 

data suggest that this enzyme may participate in the nodule 

oxygen barrier through the detoxication of ROS produced 

by high rates of respiration detected in the endodermis (indeterminate 

nodules) and in boundary layers (determinate nodules) 

(Dalton et al., 1998). 

In contrast to the preferential localization of APX in the peripheral 

layer of the nodule, the GSH and its legume-specific 

homolog hGSH are mainly present in the infected zone of 

the determinate bean nodule (Matamoros et al., 1999b). High 

content of these two thiols also occurs in the meristematic 

zone of the nodule. Moreover, the ascorbate–glutathione 

pathway was more potent in effective nodules than in ineffective 

nodules, accentuating the important protective role that 

this pathway may play for nitrogen fixation in soybean and 

alfalfa (Dalton et al., 1993). Comparable results were also observed 

in the leghemoglobin-RNA interference L. japonicus 

lines, which are altered in their nitrogen-fixation activity 

(Ott et al., 2005; Gunther et al., 2007). 

The nitrogen-fixing deficiency of S. meliloti strains mutated 

in genes belonging to redox homeostasis also indicates its 




crucial role in nitrogen-fixation metabolism. Bacterial mutants 

affected in the antioxidant defense (catalase or glutathione) 

were not only affected in their capacity to infect the plant, 

but also in their nitrogen-fixation efficiency (Jamet et al., 

2003; Harrison et al., 2005). Similarly, a peroxiredoxin (prxS)/bifunctional 

catalase–peroxidase (katG) Rhizobium etli double 

mutant has a significantly reduced nitrogen fixation capacity 

(Dombrecht et al., 2005). Additionally, S. meliloti strains mutated 

for a wide variety of cellular processes but not directly 

involved in antioxidant defense were also found to be affected 

in oxidative stress protection and symbiosis (Davies and 

Walker, 2007). Likewise, mutation of a thioredoxin-like gene 

involved in melanin production affected the response to paraquat-induced 

oxidative stress and the symbiotic nitrogen fixation 

in S. meliloti (Castro-Sowinski et al., 2007). Finally, 

a Rhizobium leguminosarum deficient in the thioredoxin-like 

cycY was unable to form nitrogen-fixing nodules on pea or 

vetch, as the mutant was defective in maturation of all c-type 

cytochromes (Vargas et al., 1994). 

The alteration of the nitrogen-fixing efficiency during senescence 

correlates with a general diminution of the antioxidant 

defense and to the detection of ROS species in the 

nodule-senescent zone. This correlation is established during 

both natural and stress-induced senescence. In this framework, 

diminution of GSH/hGSH and ASC pools was concomitant with 

a decrease in nitrogen-fixing efficiency during natural senescence 

in a large number of legumes such as soybean (Evans 

et al., 1999), pea (Groten et al., 2006), or common bean (Loscos 

et al., 2008). A similar diminution of antioxidant molecules and 

enzymes was observed in legumes submitted to various environmental 

stresses (Moran et al., 1994; Escurado et al., 1996; 

Gogorcena et al., 1997; Matamoros et al., 1999b; Loscos 

et al., 2008). The modulation of the redox balance during cell 

death and senescence is a general process in plants (Gechev 

et al., 2006; Noctor et al., 2007; Shao et al., 2008). In this framework, 

as mentioned previously, nodule senescence has a high 

degree of overlap with leaf senescence. 

One of the specific features of nodules is low oxygen pressure 

in the fixing zone to permit efficient nitrogen fixation. 

The regulation of respiration and the efficiency of the oxygen 

diffusion barrier play a crucial role in the protection of nodule 

metabolism. This regulation seems to be an active plantcontrolled 

process (Wei and Layzell, 2006). Stress-induced decrease 

of nitrogen-fixing activity is generally associated with 

a modification of the oxygen pressure in the nodule. In soybean, 

the drought and salt stresses caused a decrease in nitrogenase 

activity correlating with a diminution of nodule 

permeability (Del Castillo et al., 1994; Serraj et al., 1994). Similarly, 

soybean nodules presented modified respiration and 

intercellular space size during chilling, showing an altered 

oxygen diffusion barrier (van Heerden et al., 2008). In contrast, 

the sensitivity to salinity appears to be associated with an increase 

in nodule conductance and an increased respiration of 

nodules in M. truncatula (Aydi et al., 2004). Taken together, 

these results show that nodule efficiency is strongly affected 

by the regulation of oxygen availability that may be linked 

to ROS production. 

Another specific feature is the high carbon/nitrogen metabolism 

needed to feed the nitrogen-fixing bacteroids and to allow 

nitrogen assimilation by the plant cell. Transcriptomic 

approaches suggest that several metabolic pathways are coordinately 

up-regulated in nodules, including glycolysis, amino 

acid biosynthesis, haem, and redox metabolism (Colebatch 

et al., 2004). Glycolysis plays a primary role in feeding the bacteroids; 

environmental stresses such as salt stress, drought 

stress, and dark stress generally down-regulate the enzymes 

involved in supplying carbon to the bacteroids (Gogorcena 

et al., 1997; Galvez et al., 2005; Lopez et al., 2008). Interestingly, 

mild oxidative stress using paraquat treatment mimics 

biological modifications observed under environmental stress, 

indicating a likely involvement of redox modifications in the 

perception of environmental stress in pea nodules (Marino 

et al., 2006). In pea, this regulation of carbon metabolism 

by the cellular redox state has been recently linked to the transcriptional 

and post-translational regulation of sucrose synthase, 

the enzyme allowing sucrose assimilation in the 

nodule (Marino et al., 2008). These data underline the involvement 

of redox regulation in nodule metabolism. 

PERSPECTIVES 

The data summarized in this review clearly indicate that ROS 

and antioxidant defense play a crucial role in symbiosis between 

legumes and rhizobia. The involvement of ROS and antioxidant 

defense in key steps of the nodule formation, such as infection 

thread development and nodule meristem formation, shows 

that redox regulation is important for the nodule development. 

Moreover, several bacterial strains deficient in antioxidant defense 

exhibit an altered nitrogen-fixation capacity without 

strong modifications of their growth efficiency in free-living 

conditions, suggesting that nodule environment is more stressful 

for the bacteria. Finally, the modifications of the plant antioxidant 

defense is observed in parallel with metabolic changes 

during nodule functioning. Thus, the redox homeostasis may 

play a crucial role in the control of nodule metabolism by 

the plant. However, the fine characterization of ROS production 

and modification of the redox state during symbiosis still needs 

to be achieved. Specific protein probes sensitive to redox potential 

(Meyer et al., 2007) or to ROS accumulation (Belousov et al., 

2006) would be useful tools for realizing the spatiotemporal 

analysis of H 2O 2 production and redox changes. Furthermore, 

taking into account that plastids are involved in GSH synthesis 

(Pasternak et al., 2008) and in retrograde signaling (Nott et al., 

2006), it would be worth addressing the role of these organelles 

in redox homeostasis. The development of two legume model 

systems, M. truncatula (www.medicago.org/) and L. japonicus 

(www.lotusjaponicus.org/) will facilitate an efficient analysis 

of the redox-dependent metabolism. 

Legume–rhizobia symbiosis is an interesting model for 

studying compatible plant–microorganism interactions and 



plant development processes. Thus, information obtained 

with this model will be of general interest for plant biologists. 

From a more pragmatic point of view, legumes are unique, as 

they do not need nitrogen-fertilizers (which need fossil energy 

for their production) and present high protein content. Thus, 

legumes have a crucial role to play in the development of more 

sustainable and multifunctional agro-ecosystems. 

FUNDING 

This work is funded by CNRS, INRA and the University of Nice- 

Sophia Antipolis. The Agence Nationale de la Recherche is supporting 

this work through the RHYTHMS project (contract number: 

BLAN07-2_182872). 

ACKNOWLEDGMENTS 

We gratefully acknowledge Julie Hopkins, Erica Stein, and Ryan 

Gutierrez for critical reading of the manuscript. C.C. is the recipient 

of a post-doctoral fellowship from the ‘Ministère de l’Enseignement 

Supérieur et de la Recherche’. I.D. is the recipient of 

a post-doctoral fellowship from the CNRS. We apologize to our colleagues 

whose works were not cited here. No conflict of interest 

declared. 

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Redox Changes during the Legume–Rhizobium Symbiosis

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