Journal of Integrative Plant Biology 2010, 52 (2): 195–204

Invited Expert Review

Reactive Oxygen Species during Plant-microorganism
Early Interactions
∗ ∗ ∗∗
Amrit K. Nanda1 , Emilie Andrio2 , Daniel Marino2 , Nicolas Pauly2 and Christophe Dunand1
1 SCSV-UMR5546 CNRS/UPS, 24 Chemin de Borderouge, BP 42617 Auzeville, 31326 Castanet-Tolosan, France
2 InteractionsBiotiques et Santé Végétale, UMR INRA 1301 – Université de Nice-Sophia Antipolis – CNRS 6243, 400 Route des Chappes,
BP167, F-06903 Sophia Antipolis Cedex, France
∗
These authors contribute equally to this work
∗∗
Corresponding author
Tel: +33 5 6219 3557; Fax: +33 5 6219 3502; E-mail: dunand@scsv.ups-tlse.fr
Available online on 5 February 2010 at www.jipb.net and www.interscience.wiley.com/journal/jipb
doi: 10.1111/j.1744-7909.2010.00933.x

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 signalling molecules involved in several developmental
processes in all organisms. Previous studies have clearly shown
that an oxidative burst often takes place at the site of attempted
invasion during the early stages of most plant-pathogen interac-
tions. Moreover, a second ROS production can be observed during
certain types of plant-pathogen interactions, which triggers hyper-
sensitive cell death (HR). This second ROS wave seems absent
Christophe Dunand during symbiotic interactions. This difference between these two
(Corresponding author) responses is thought to play an important signalling role leading
to the establishment of plant defense. In order to cope with the
deleterious effects of ROS, plants are fitted with a large panel
of enzymatic and non-enzymatic antioxidant mechanisms. Thus,
increasing numbers of publications report the characterisation of ROS producing and scavenging
systems from plants and from microorganisms during interactions. In this review, we present the current
knowledge on the ROS signals and their role during plant-microorganism interactions.

Nanda AK, Andrio E, Marino D, Pauly N, Dunand C (2010) Reactive oxygen species during plant-microorganism early interactions. J. Integr. Plant
Biol. 52(2), 195–204.

stomata closing (Pei et al. 2000), plant-pathogen interactions

Introduction
Reactive Oxygen Species (ROS), such as superoxide anion
r
(O 2 − ) and hydrogen peroxide (H 2 O 2 ), are by-products con-
stantly produced during normal metabolic processes, such
as photosynthesis or glycolysis. ROS produced at high
levels have first been described as lethal for the cell integrity.
However, high ROS production is also necessary for plant
defense (oxidative burst, necrosis). Currently, their involve-
ment as signal molecule during cellular growth, control of
(Apel and Hirt 2004), programmed cell death (Gechev and
Hille 2005) and stress responses have been demonstrated
in plants (Laloi et al. 2007; Miller et al. 2007). The regu-
lation and the involvement of ROS in Legume – Rhizobia
symbiotic relations (Santos et al. 2001; Ramu et al. 2002;
Shaw and Long 2003; Rubio et al. 2004) and during the
establishment of both endo- and ectomycorrhiza have also
been described (Fester and Hause 2005; Baptista et al.
2007).


C 2010 Institute of Botany, Chinese Academy of Sciences
196 Journal of Integrative Plant Biology Vol. 52 No. 2 2010

In both symbiotic and pathogenic relations, the transient
production of ROS is detected in the early events of plant-
microorganism interactions and appears as the only common
feature of the plant responses. This production called oxidative
burst could be considered as a specific signal during the
interaction process.
Prevention of ROS toxicity and control of ROS signalling
require a large gene network, the so called “ROS gene network”
is composed of at least 150 genes in Arabidopsis (Mittler et al.
2004). A smaller network is also detected in microorganisms
(Passardi et al. 2007). Several families of proteins from plants
and microorganisms are associated with the regulation of ROS
levels (Table 1). Among them, catalases (Kat), detected in all
kingdoms, and catalase-peroxidases (CP), present in some
fungi and in the majority of bacteria, can both reduce H 2 O 2
(Passardi et al. 2007). Other proteins detected in all kingdoms
can generate ROS such as NADPH oxidases (NOx/RBOH), or
scavenge ROS such as glutathione peroxidases (GPx) (Margis
et al. 2008), peroxiredoxins (Rouhier and Jacquot 2002) and
thioredoxins (Alkhalfioui et al. 2008). The plant specific Class
III peroxidases (Prx) are also members of the “ROS gene
network” and have the interesting capacity to both scavenge
and produce ROS (Passardi et al. 2004).
This review describes the involvement of ROS and highlights
the different ROS producing and ROS scavenging enzymatic
systems characterized during plant biotic interactions. Special
attention will be paid to plant symbiotic interactions. In the
meantime, there are several recent specialised reviews where
the reader can find more information about the role of ROS
during plant abiotic stresses (Miller et al. 2008; Torres and
Dangl 2005).
ROS Accumulation during
Plant-microorganism Interactions
During the first minutes of interaction between plants and
microorganisms, a molecular dialogue involving several signal
molecules, takes place in the rhizosphere and at the cell
surface, leading to physical interaction. For example, in the
case of the Legume – Rhizobia symbiotic interaction, flavonoids
from the plant root exudates induce the synthesis of Nodulation
Factor (NF) from Rhizobia. Both compounds are responsible for
the setup of the early interaction steps and for the establishment
of the new root organ, the nodule (for review, Oldroyd and
Downie 2008). A similar dialogue is observed during mycor-
rhizal fungus and plant interaction leading to the production
of plant strigolactons (Akiyama et al. 2005) and putative Myc
factor by the fungus (Kosuta et al. 2003).
Plant-pathogen interactions
One of the most rapid defense reactions to pathogen at-
tack is the so-called oxidative burst, which constitutes to the
r
ROS production, primarily superoxide (O 2 − ) and hydrogen
peroxide (H 2 O 2 ), at the site of attempted invasion (Apel and
Hirt 2004). This response is involved in pathogenic as well
as in symbiotic interactions. Doke first reported the oxidative
burst (Doke 1983), demonstrating that potato tuber tissue
r
generated O 2 − that is rapidly transformed into H 2 O 2 following
inoculation with an avirulent oomyceta Phytopthera infestans.
Similar H 2 O 2 production is also observed during avirulent
interaction between the bacteria Pseudomonas syringae strain
DC3000 and Arabidopsis (Alvarez et al. 1998). A virulent

Table 1. ROS scavenging and producing protein family distribution across the major kingdoms
Prokaryotes Plants Fungi Other eukaryotes
√ √ √ √
Animal peroxidase (12 subfamilies)
√ √ √ √
Catalase (Kat)
√
Di-haem peroxidase superfamily
Class I peroxidase:
√ √
Ascorbate peroxidase (APx)
√ ∗ ∗
Catalase peroxidase (CP)
√ √
Cytochrome C peroxidase (CcP)
√
Class III peroxidase
Thioredoxin (TRX)-like superfamily
√ √ √ √
Glutathione peroxidase (GPx)
√ √ √ √
Peroxiredoxin
√ √ √ √
Other Thioredoxin (AhpC/TSA. . .)
√ √ √
NADPH oxidase
√
Organic hydroperoxide resistance (ohr) – thiol-dependent peroxidase
√ √ √ √
Superoxide Dismutase (SOD)
The presence or the absence of the different families in the various taxonomic groups has been identified. ∗ presence due to lateral gene
transfer.

r
race of the same pathogen failed to induce O 2 − produc-
r
tion. Subsequently, O 2 − generation has been identified in a
wide range of plant-pathogen interactions involving avirulent
bacteria, fungi, and viruses (Low and Merida 1996). Since
then, further research has shown that avirulent pathogens
induce a biphasic ROS production in plants, consisting of a
low amplitude first phase, followed by a much higher and
sustained accumulation during the second phase (Lamb and
Dixon 1997; Torres et al. 2006). However, only the first phase
has been detected during interactions with virulent pathogens
(Bolwell et al. 2002). Furthermore, in the case of symbiotic
interactions, ROS have also been observed but a suppression
of the second wave of ROS seems to take place (Shaw and
Long 2003; Lohar et al. 2007). This second response or lack
of response is thought to play an important signalling role
in the activation of plant defense. ROS have therefore been
proposed to play a key role in the establishment of plant
defense responses (Levine et al. 1994). In fact, the important
ROS accumulation during the second phase has been reported
to precede the hypersensitive response (HR) cell death that
often accompanies successful pathogen recognition leading
to the incompatible interaction (Mehdy 1994; Levine et al.
1996).
These events establish the involvement of ROS signalling in
the activation or deactivation of the plants defense processes
during different plant-microorganism interactions.
Legume – Rhizobia symbiotic interactions
The symbiosis between legumes and compatible Rhizobia
takes place in a nitrogen-limited environment thanks to a
molecular dialogue between the two actors. Rhizobia secret
Nod Factors (NFs) in response to plant root exudates con-
taining flavonoids. The perception of these NFs by the plant
triggers several responses such as ion changes, cytoplasmic
alkanisation, calcium oscillations and gene expression leading
to the formation of an infection thread and a new organ, the
root nodule, containing the nitrogen fixing rhizobia bacteroid
(Cardenas et al. 2000; Oldroyd and Downie 2004). The plant
provides bacteria with energy and a micro-aerobic environment
compatible with nitrogenase activity. In exchange, bacteria
provide the plant with a nitrogen supply. Nodules represent
therefore a unique model for the study of developmental
processes, plant-microorgansim and carbon/nitrogen/oxygen
metabolism interactions.
The involvement of ROS during the Legume – Rhizobium
symbiosis has been highlighted during this last decade (Pauly
et al. 2006). For example, during the establishment of the
symbiotic interaction, by using Nitroblue tetrazolium (NBT) that
r
forms a dark blue precipitate with O 2 − (Bielski et al. 1980),
ROS – Destructive or Signalling Molecules 197
r
can be detected in infection threads, indicating that O 2 − is
produced during the infection process and could have a role
in the control of the bacteria development (Santos et al. 2001;
Ramu et al. 2002). ROS production in the infection threads is
dependent on the NFs production because no ROS was ob-
served when Medicago truncatula plants were inoculated with
bacteria unable to produce NFs (Ramu et al. 2002). Recently,
Jamet et al. (2007) showed that an S. meliloti mutant impaired
in H 2 O 2 steady state is affected in its ability to establish an
optimal symbiosis. This clearly indicates the role of H 2 O 2 in
the early steps of the interaction (Jamet et al. 2007). Moreover,
the generation of ROS in the cortical cells of M. truncatula
roots after inoculation with Sinorhizobium meliloti was observed
in vivo, using a ROS fluorescent probe (Peleg-Grossman et al.
2007). Moreover, a transient increase in intracellular ROS level
at the tip of growing Phaseolus vulgaris root hairs has been
shown within a few minutes after treatment with NFs (Cardenas
et al. 2008). However, the extracellular ROS situation may be
different in the very early steps of the symbiotic interaction,
where the production of H 2 O 2 appears to be inhibited by NFs
treatment (Shaw and Long 2003; Lohar et al. 2007) or at least
not induced. In the same way, a S. meliloti nodC− mutant,
defective in NFs biosynthesis, triggers an important increase in
H 2 O 2 accumulation in Medicago sativa roots after inoculation
(Bueno et al. 2001). Moreover, the compatible interaction
between M. sativa and S. meliloti is linked, at least in part, with
an increase of the antioxidant defense (particularly catalase
and lipoxygenase) during the preinfection period (Bueno et al.
2001).
More recently, the role of ROS in root hair deformation in
the M. truncatula – S. meliloti symbiosis has been highlighted
(Lohar et al. 2007). Exogenous application of ROS as well
as the inhibition of ROS production using diphenylene iodo-
nium (DPI), a commonly used NADPH oxidase inhibitor, both
prevented root hair swelling and branching normally induced
following treatment with NFs. However, transient treatment
of M. truncatula roots with DPI, mimicked NFs treatment
and resulted in root hair branching in the absence of NFs.
Interestingly, the same transient DPI treatment on non-legumes
such as Arabidopsis thaliana and Lycopersicon esculentum did
not induce root hair branching. These results suggest a role
for the transient reduction of ROS accumulation in governing
NF-induced root hair deformation in legumes (Lohar et al.
2007).
The transient decrease of intracellular ROS accumulation in
legume root hairs, in response to rhizobial secretion of NFs,
seems to play a key role in a compatible Legume-Rhizobium
interaction by actively promoting the root infection by bacteria.
However, without recognition of the NFs or by using non host
NFs, the plant seems to consider the bacteria as a pathogen
and mobilizes its defense mechanisms.
198 Journal of Integrative Plant Biology Vol. 52 No. 2 2010

Plant-fungus symbiotic interactions r

as SOD convert O 2 − to H 2 O 2 and Kat convert H 2 O 2 to water.
The combined action of these two enzymes seems play an
Most land plants can form mutualistic symbiosis with myc-
important role during plant-EM fungus interactions (Baptista
orrhizal fungi which can be divided into two categories: ec-
et al. 2007).
tomycorrhizae (EM) with extracellular hyphal structures and
Once again, ROS seem to play an important role in the
endomycorrhizae or arbuscular mycorrhizae (AM) with intra-
different symbiotic interactions. These results suggest a main
cellular hyphal structures (Bonfante and Anca 2009). In AM
role in the control of the fungus proliferation in the plant.
symbioses, fungal hyphae form appressoria at the root surface,
However, knowledge about ROS during early symbiotic inter-
before intercellular invasion of epidermal and root cortical
actions between plants and fungi is very limited, due to the
cells (Harrison 2005). Intensive nutrient exchange takes place
asynchronous nature of the mycorrhization process.
across membrane interfaces between the fungus and the plant
during symbiosis. The fungus provides the plant with nutrients
like phosphorus, which the plant can have difficulty extracting
Scavenging and ROS Producing Systems
from the soil. In return, the plant delivers carbon and lipids to
during Plant-microorganism Interactions
the fungal symbionts.
As observed during the other plant-microorganism interac- ROS are known to play major roles in various plant and mi-
tions, ROS have also been evidenced in mycorrhizal symbiosis: croorganism developmental processes, such as cell elongation
in the M. truncatula – Glomus intraradices interaction, H 2 O 2 (root hairs, pollen tube or appressoria growth) and during
accumulation observed in plant cells was hypothesized to be biotic interactions (Figure 1). In order to avoid ROS accu-
a consequence of activation of a plant plasma membrane mulation leading to cell death (Mittler et al. 2004) organisms
NADPH oxidase (NOx) in response to the fungus (Salzer et al. have evolved enzymatic and non-enzymatic antioxidant mech-
1999), analogous to what occurs during the HR. In this case, anisms constantly generating and deteriorating ROS (Figure 1).
H 2 O 2 accumulation is mostly observed in arbuscule-containing In plants, ROS are unavoidable by-products of biochemical
cells, more precisely surrounding the arbuscular structures. pathways, such as glycolysis and photosynthesis. As a result,
This suggests that ROS play a role in the control of fungal plants have evolved enzymatic and non-enzymatic antioxidant
proliferation within the plant (Fester and Hause 2005). How- mechanisms to eliminate ROS and avoid oxidative destruction
ever, the involvement of ROS during the early plant-AM fungus (Apel and Hirt 2004). On the other hand, ROS production is
interactions has yet to be studied. Recently, H 2 O 2 production necessary for cell elongation (root hairs, appresoria growth)
was also reported during the symbiosis between Gigaspora and plant-microorganism interactions. It is therefore necessary
margarita and two legume species including M. truncatula for the plant to possess very complex and well-tuned ROS
and Lotus japonicus, mainly located in the intraradical fungal producing and scavenging systems capable of maintaining
structures (Lanfranco et al. 2005). ROS homeostasis in the cells.
Evidence for the participation of ROS and antioxidant sys-
tems in ectomycorrhizal symbiosis has been found between
the fungus Pisolithus tinctorius and the plant Castanea sativa
(Baptista et al. 2007). During the early stages of this symbiosis,
three peaks of H 2 O 2 production were detected in C. sativa 2 h,
r
5 h and 11 h post inoculation, the first two coinciding with O 2 −
r −
bursts. It is noteworthy that no O 2 was detected by NBT stain-
r
ing in P. tinctorius hyphae. The first phase of production of O 2 −
seems to be extracellular which suggests that the early EM
fungus-plant interaction takes place at the cell wall and plasma
membrane surfaces (Baptista et al. 2007). This first phase
is similar to pathogenic attack responses, where the main
sources of ROS production have been identified as membrane-
bound NOx (Wojtaszek 1997). During the second phase of
r
ROS production, O 2 − accumulates in microdomains within
cells. This could result from activation of the ROS-producing
systems and down-regulation of the ROS-scavenging ones.
Furthermore, superoxide dismutases (SOD) appear to be up- Figure 1. Schematic representation of dual ROS level regula-
regulated and catalases (Kat) to be down-regulated during tion (production and scavenging) and the main ROS functions
these early stages. This could explain the H 2 O 2 accumulation, related to plant-microorganism interactions.
 ROS – Destructive or Signalling Molecules 199

Figure 2. ROS regulation during development and plant-microorganism interactions.

Constitutive ROS production (photosynthesis and respiration) have also been included in the cartoon. T-bars and arrows correspond to
scavenging and production of ROS respectively. Pink lines stand for ROS involved in plant-microorganism interactions and blue lines for
ROS involved in the development

We now pay special attention to ROS regulating systems
during plant-microorganism interactions.
Enzymatic ROS scavenging mechanisms involved in
plant-microorganism interactions
The large battery of ROS scavenging enzymes included in
the “ROS gene network” contains catalases (Kat), superoxide
dismutases (SOD), ascorbate peroxidases (APx, detected in
plants) cytochrome C peroxidases (CcP, detected in fungi).
They are present in several intracellular compartments as well
as in the apoplast in order to regulate both intracellular and
extracellular ROS accumulation (Mittler et al. 2004). Consider-
ing the extracellular region, both plant and microorganism are
capable of regulating the ROS level in this area during the early
steps of the interaction (Figure 2).
The symbiotic Rhizobia appears to have an efficient an-
tioxidant defense. Indeed, although ROS were present in the
infection threads they weren’t detected inside the bacteria
progressing within the infection thread (Santos et al. 2001;
Rubio et al. 2004). Indeed, S. meliloti possesses two SOD that
r
convert O 2 − to O 2 and H 2 O 2 (Santos et al. 2000; Hérouart
et al. 2002) and three heme b containing catalases, which are
able to scavenge H 2 O 2 (Hérouart et al. 1996; Ardissone et al.
2004). Bacterial catalases appear to play an important role
in the nodule formation process as the double katB/katC and
200 Journal of Integrative Plant Biology Vol. 52
katA/katC mutants of S. meliloti are strongly impaired in nodule
formation (Jamet et al. 2003). In addition to the catalases, the
S. meliloti genome contains three thiol peroxidase encoding
genes: the alkyl hydroperoxide reductase ahpC-like and two
organic hydroperoxide resistance ohr-like genes. Both types
of enzyme display biochemically equivalent functions and cat-
alyze the reduction of organic peroxides to the corresponding
less toxic organic alcohols.
In the case of a pathogenic interaction between rice and
Magnaporthe griseae, the causal agent of rice blast disease,
the fungus has to overcome the plants innate immunity in
order to infect it. The massive production of ROS during the
early stages of interaction is part of the plants innate immunity
response. To overcome this line of defense, M. griseae must
be able to counter the oxidative burst by producing ROS
scavenging enzymes. A novel gene related to pathogenecity
has recently been isolated in M. griseae: Defense Suppressor 1
(DES1) (Chi et al. 2009) . des1 deficient mutants were hyper-
sensitive to exogenous oxidative stress and the transcription
of extracellular enzymes such as peroxidases and laccases
were severely reduced. In interaction with a susceptible rice
cultivar, the mutants displayed an important reduction of infec-
tious hyphal extension, leading to a decrease in pathogenic-
ity. Interestingly, the des1 deficient mutants recovered their
normal infectious growth when interacting with DPI treated
plant tissue. These results strongly support the possibility that
ROS play a major role in the first line of plant defenses at
the cell surface both as toxic molecules as well as signalling
actors.
Mechanisms to generate ROS
Several different enzymes have been implicated in the genera-
tion of ROS. Among these, NADPH oxidases (NOx) correspond
to one of the most studied systems that play an important role in
the production of superoxide radicals during the oxidative burst
to defend cells from invasion. NOx are integral membrane pro-
teins capable of oxidizing NADPH in the cell as well as reducing
molecular oxygen into superoxide radicals in the apoplast
(Sumimoto 2008), which is quickly dismutated into H 2 O 2 either
spontaneously or by SOD enzymes. ROS produced by the
NADPH oxidases function in defense, development and redox-
dependent signalling. They share common structural features
and are evolutionarily of ancient origin and thus ubiquitous
in multicellular eukaryotes (Bedard and Krause 2007; Bedard
et al. 2007). In plants, NADPH oxidases form a small multigenic
family and are involved in diverse events including innate
immunity development. Due to the fact that ROS are toxic and in
many cases short-lived, the activity of these oxidases is tightly
regulated both temporally and spatially.
The use of DPI, that inhibits flavoproteins such as NOx,
and abolished ROS production, strongly supports the possible
No. 2 2010
involvement of M. truncatula NOx homologues in ROS pro-
duction. Moreover, a DPI treatment during the early stages
of M. truncatula – S. meliloti interaction not only abolished
ROS production but also suppressed root hair curling and infec-
tion thread formation (Peleg-Grossman et al. 2007; Cardenas
et al. 2008). These results emphasize the involvement of M.
truncatula NADPH oxidase homologues in the early steps of
Rhizobium infection.
The involvement of plant NOx in plant-microorganism inter-
actions have clearly been shown (see earlier reviews Apel and
Hirt 2004; Torres and Dangl 2005). NOx are present in all the
fungi forming fruit bodies where they seem to participate in
sexual reproduction. The inactivation of Aspergillus nidulans
NoxA gene resulted in a decrease of ROS production, inhibition
of the formation of cleistothecia at early stages of development,
stimulation of mycelium growth and suppression of asexual
reproduction (Lara-Ortiz et al. 2003). In addition NOx from fungi
are also important during the infection process. Moreover in
the symbiotic interaction between the fungus Epistle fistulae
and the plant Folium perenne, a NOXA deficient fungus mutant
is unable to undergo symbiosis and induces plant death. This
shows that fungus produced ROS that also play a major role
in the establishment of this symbiosis (Takemoto et al. 2006;
Tanaka et al. 2006).
More recently, several reports from the microbe side indicate
a major role of these genes in the pathogenicity process (Egan
et al. 2007; Giesbert et al. 2008) and thus, play a positive role
for the pathogen. Accordingly, during plant infection, NOx from
M. grisea generate ROS. This oxidative burst is associated
with the development of specialised infection structures called
appressoria. Pharmacological scavenging of these oxygen rad-
icals significantly delayed the development of appressoria and
affected their morphology. Using a genetic approach targeting
two NOx genes, Egan et al. (2007) showed that these genes are
independently required for pathogenicity of M. grisea (inability
to initiate appressorium-mediated cuticle penetration for the
mutants) and are involved in ROS production (Egan et al.
2007). In a similar approach, the deletion of a putative NOx
from the ergot fungus of ryegrass, Claviceps purpurea, has an
impact on germination of conidia and pathogenicity, although its
involvement in focusing ROS production has not been shown
(Giesbert et al. 2008).
Other proteins, such as class III peroxidases, regulate for
ROS homeostasis. Class III peroxidases are only detected in
Viridiplantae and are present as large multigenic families in
all land plants (Bakalovic et al. 2006). Released from the cell
surface into the apoplast, peroxidases are an important class
of enzymes responsible for the stress-induced formation and
degradation of ROS (Bolwell et al. 2002; Bindschedler et al.
2006; Fecht-Christoffers et al. 2006). Apart from their indirect
role in H 2 O 2 detoxification through its peroxidative activities,
some apoplastic class III peroxidases can also generate O 2 −
r

or H 2 O 2 at physiological pHs via its oxidative cycle (Minibayeva
et al. 2009). The cell wall has an enormous capacity to retain
proteins in normal growth conditions, most of the peroxidases
for instance, which may be released following abiotic stress.
The involvement of class III peroxidases during the symbiotic
process has already been observed. For example, Rip1, en-
coding a peroxidase from Medicago is rapidly and transiently
induced by Rhizobium meliloti or after NFs treatment (Cook
et al. 1995). Moreover, this gene is induced by H 2 O 2 (Ramu
et al. 2002).
More recently, a class III peroxidase (Srprx1) has been
shown to be crucial for the bacterial invasion of the tropical
Legume, Sesbania rostrata (Den Herder et al. 2007). The ex-
pression of Srprx1 is strictly dependent on bacterial nodulation
factors (NFs) and could be modulated by H 2 O 2 , a downstream
signal for crack-entry invasion. Its expression was not induced
after wounding or pathogen attack, indicating that the peroxi-
dase is a symbiosis-specific isoform. More interestingly, lack of
Srprx1 gene expression could cause an aberrant structure of
the infection threads (Den Herder et al. 2007).
OsPrx53, encoding a peroxidase from rice, is the strongest
gene induced after Glomus infection (Guimil et al. 2005).
Peroxidases seem important for the initiation of symbiosis but
no direct evidence has demonstrated their implication for ROS
production in the early steps of interaction and the development
of the infection (Guimil et al. 2005).
Furthermore, the involvement of peroxidases in H 2 O 2
synthesis during plant-pathogen interactions has been re-
cently highlighted (Choi et al. 2007). Thus, H 2 O 2 produc-
tion is also compromised after inoculation of Capsicum an-
num, silenced for a peroxidase, by avirulent Xanthomonas
campestris bacteria (Choi et al. 2007). This clearly demonstrate
the peroxidase involvement in ROS production (Choi et al.
2007).
Although NOx and peroxidases represent the main charac-
terized plant ROS producing systems at the plant cell surface,
one should note that several oxidative and reductive systems
are present in the plant plasmalemma (Vuletic et al. 2005).
More interestingly, special attention has been recently paid to
the plasma membrane microdomains. Recently, a proteomic
approach based on the purification of lipid-rafts in plasma
membrane from M. truncatula identified several putative ROS
producing systems, including peroxidase (Furt et al. 2007;
Lefebvre et al. 2007). This approach does not allow the iden-
tification of NOx although their presence has been previously
shown in elicitor treated tobacco cells (Mongrand et al. 2004).
Other possible sources for H 2 O 2 in the Legume – Rhizobium
symbiosis are germin-like oxalate oxidases or diamine oxi-
dases (Wisniewski et al. 2000). Indeed, a germin-like oxidase
from Pisum sativum has been characterized (PsGER1). This
protein has a superoxide dismutase activity, and is associated
with nodules (Gucciardo et al. 2007).
ROS – Destructive or Signalling Molecules 201
Conclusions
ROS are produced by all living organisms, either constitu-
tively as by-products of several metabolic processes or in
a more controlled manner during developmental processes
as well as at the early stages of plant-microorganism in-
teractions. The oxidative burst, that often takes place at
the very first stage of the interaction, acts as the first line
of plant defense. The microbe must therefore produce ROS
scavenging enzymes in order to successfully infect the plant
or down-regulate the plant ROS producing systems. This
massive ROS production and scavenging takes place in the
apoplast between the cell surfaces of the two organisms
or in the rhizosphere. However, the oxidative burst seems
to differ in intensity and length between plant-pathogen
and plant-symbiote interactions. This difference could act
as a specific signal predefining the host’s response to the
microbe.
Moreover, the plant NOx have been found to play an
important role in the oxidative burst during plant-pathogen
interactions. However, no functional evidence of NOx in-
volvement in ROS production during Legume – Rhizobium
interactions has been found. This further supports the idea
that different ROS regulating systems could be activated
by interactions with a pathogen or a symbiote. In this line,
class III peroxidases could be a good candidate for ROS
regulation.
Whatever the system involved during the different sym-
biotic interactions, it would be of interest to analyze the
consequences of modifying plant ROS-producing activities
on the symbiotic capacities. This could allow us to better
understand the signalling role of ROS molecules and it’s
consequences on the establishment of symbiosis.
Acknowledgments
We thank Julie Hopkins for her critical reading of the manuscript
and for her help for the English editing.
Received 1 Sept. 2009 Accepted 29 Dec. 2009
References
Akiyama K, Matsuzaki K, Hayashi H (2005) Plant sesquiterpenes
induce hyphal branching in arbuscular mycorrhizal fungi. Nature
435, 824–827.
Alkhalfioui F, Renard M, Frendo P, Keichinger C, Meyer Y, Gelhaye
E, Hirasawa M, Knaff DB, Ritzenthaler C, Montrichard F (2008)
202 Journal of Integrative Plant Biology Vol. 52
A novel type of thioredoxin dedicated to symbiosis in legumes. Plant
Physiol. 148, 424–435.
Alvarez ME, Pennell RI, Meijer PJ, Ishikawa A, Dixon RA, Lamb C
(1998) Reactive oxygen intermediates mediate a systemic signal
network in the establishment of plant immunity. Cell 92, 773–784.
Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative
stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373–399.
Ardissone S, Frendo P, Laurenti E, Jantschko W, Obinger C,
Puppo A, Ferrari RP (2004) Purification and physical-chemical
characterisation of the three hydroperoxidases from the symbiotic
bacterium Sinorhizobium meliloti. Biochemistry 43, 12692–12699.
Bakalovic N, Passardi F, Ioannidis V, Cosio C, Penel C, Falquet L,
Dunand C (2006) PeroxiBase: a class III plant peroxidase database.
Phytochemistry 67, 534–539.
Baptista P, Martins A, Pais MS, Tavares RM, Lino-Neto T (2007)
Involvement of reactive oxygen species during early stages of ecto-
mycorrhiza establishment between Castanea sativa and Pisolithus
tinctorius. Mycorrhiza 17, 185–193.
Bedard K, Krause KH (2007) The NOX family of ROS-generating
NADPH oxidases: physiology and pathophysiology. Physiol. Rev.
87, 245–313.
Bedard K, Lardy B, Krause KH (2007) NOX family NADPH oxidases:
not just in mammals. Biochimie 89, 1107–1112.
Bielski BHJ, Shine GG, Bajuk S (1980) Reduction of nitroblue tetra-
zolium by CO 2 − and O 2
r− radicals. J. Phys. Chem. 84, 830–833.
Bindschedler LV, Dewdney J, Blee KA, Stone JM, Asai T, Plotnikov
J, Denoux C, Hayes T, Gerrish C, Davies DR, Ausubel FM, Bol-
well GP (2006) Peroxidase-dependent apoplastic oxidative burst in
Arabidopsis required for pathogen resistance. Plant J. 47, 851–863.
Bolwell GP, Bindschedler LV, Blee KA, Butt VS, Davies DR, Gard-
ner SL, Gerrish C, Minibayeva F (2002) The apoplastic oxidative
burst in response to biotic stress in plants: a three-component
system. J. Exp. Bot. 53, 1367–1376.
Bonfante P, Anca IA (2009) Plants mycorrhizal fungi, and bacteria: a
network of interactions. Annu. Rev. Microbiol. 63, 363–383.
Bueno P, Soto MJ, Rodriguez-Rosales MP, Sanjuan J, Olivares
J, Donaire JP (2001) Time-course of lipoxygenase, antioxidant
enzyme activities and H 2 O 2 accumulation during early stages of
Rhizobium legume symbiosis. New Phytol. 152, 91–96.
Cardenas L, Holdaway-Clarke TL, Sanchez F, Quinto C, Feijo JA,
Kunkel JG, Hepler PK (2000) Ion changes in legume root hairs
responding to Nod factors. Plant Physiol. 123, 443–452.
Cardenas L, Martinez A, Sanchez F, Quinto C (2008) Fast, transient
and specific intracellular ROS changes in living root hair cells
responding to Nod factors (NFs). Plant J. 56, 802–813.
Chi MH, Park SY, Kim S, Lee YH (2009) A novel pathogenicity gene
is required in the rice blast fungus to suppress the basal defenses
of the host. PLoS Pathog. 5, e1000401.
Choi HW, Kim YJ, Lee SC, Hong JK, Hwang BK (2007) Hydrogen
peroxide generation by the pepper extracellular peroxidase CaPO 2
activates local and systemic cell death and defense response to
bacterial pathogens. Plant Physiol. 145, 890–904.
No. 2 2010
Cook D, Dreyer D, Bonnet D, Howell M, Nony E, VandenBosch
K (1995) Transient induction of a peroxidase gene in Medicago
truncatula precedes infection by Rhizobium meliloti. Plant Cell 7,
43–55.
Den Herder J, Lievens S, Rombauts S, Holsters M, Goormachtig S
(2007) A symbiotic plant peroxidase involved in bacterial invasion of
the tropical legume Sesbania rostrata. Plant Physiol. 144, 717–727.
Doke N (1983) Involvement of superoxide anion generation in hy-
persensitive response of potato tuber tissues to infection with an
incompatible race of Phytophthora infestans. Physiol. Plant Pathol.
23, 345–347.
Egan MJ, Wang ZY, Jones MA, Smirnoff N, Talbot NJ (2007)
Generation of reactive oxygen species by fungal NADPH oxidases
is required for rice blast disease. Proc. Natl. Acad. Sci. USA 104,
11772–11777.
Fecht-Christoffers MM, Fuhrs H, Braun HP, Horst WJ (2006)
The role of hydrogen peroxide-producing and hydrogen peroxide-
consuming peroxidases in the leaf apoplast of cowpea in man-
ganese tolerance. Plant Physiol. 140, 1451–1463.
Fester T, Hause G (2005) Accumulation of reactive oxygen species in
arbuscular mycorrhizal roots. Mycorrhiza 15, 373–379.
Furt F, Lefebvre B, Cullimore J, Bessoule JJ, Mongrand S (2007)
Plant lipid rafts: fluctuat nec mergitur. Plant Signal. Behav. 2, 508–
511.
Gechev TS, Hille J (2005) Hydrogen peroxide as a signal controlling
plant programmed cell death. J. Cell Biol. 168, 17–20.
Giesbert S, Schurg T, Scheele S, Tudzynski P (2008) The NADPH
oxidase CpNOX1 is required for full pathogenicity of the ergot fungus
Claviceps purpurea. Mol. Plant Pathol. 9, 317–327.
Gucciardo S, Wisniewski JP, Brewin NJ, Bornemann S (2007)
A germin-like protein with superoxide dismutase activity in pea
nodules with high protein sequence identity to a putative rhicadhesin
receptor. J. Exp. Bot. 58, 1161–1171.
Guimil S, Chang HS, Zhu T, Sesma A, Osbourn A, Roux C,
Ioannidis V, Oakeley EJ, Docquier M, Descombes P, Briggs SP,
Paszkowski U (2005) Comparative transcriptomics of rice reveals
an ancient pattern of response to microbial colonization. Proc. Natl.
Acad. Sci. USA 102, 8066–8070.
Harrison MJ (2005) Signaling in the arbuscular mycorrhizal symbiosis.
Annu. Rev. Microbiol. 59, 19–42.
Hérouart D, Baudouin E, Frendo P, Harrison J, Santos R, Jamet
A, Van de Sype G, Touati D, Puppo A (2002) Reactive oxygen
species, nitric oxide and glutathione : a key role in the establishment
of the legume – Rhizobium symbiosis. Plant Physiol. Biochem. 40,
619–624.
Hérouart D, Sigaud S, Moreau S, Frendo P, Touati D, Puppo A
(1996) Cloning and characterization of the katA gene of Rhizobium
meliloti encoding a hydrogen peroxide-inducible catalase. J. Bacte-
riol. 178, 6802–6809.
Jamet A, Mandon K, Puppo A, Hérouart D (2007) H 2 O 2 is required for
optimal establishment of the Medicago sativa/Sinorhizobium meliloti
symbiosis. J. Bacteriol. 189, 8741–8745.

Jamet A, Sigaud S, Van de Sype G, Puppo A, Hérouart D (2003)
Expression of the bacterial catalase genes during Sinorhizobium
meliloti-Medicago sativa symbiosis and their crucial role during the
infection process. Mol. Plant Microbe Interact. 16, 217–225.
Kosuta S, Chabaud M, Lougnon G, Gough C, Denarie J, Barker DG,
Becard G (2003) A diffusible factor from arbuscular mycorrhizal
fungi induces symbiosis-specific MtENOD11 expression in roots of
Medicago truncatula. Plant Physiol. 131, 952–962.
Laloi C, Stachowiak M, Pers-Kamczyc E, Warzych E, Murgia I,
Apel K (2007) Cross-talk between singlet oxygen- and hydrogen
peroxide-dependent signaling of stress responses in Arabidopsis
thaliana. Proc. Natl. Acad. Sci. USA 104, 672–677.
Lamb C, Dixon RA (1997) The oxidative burst in plant disease
resistance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 251–275.
Lanfranco L, Novero M, Bonfante P (2005) The mycorrhizal fungus
Gigaspora margarita possesses a CuZn superoxide dismutase that
is up-regulated during symbiosis with legume hosts. Plant Physiol.
137, 1319–1330.
Lara-Ortiz T, Riveros-Rosas H, Aguirre J (2003) Reactive oxygen
species generated by microbial NADPH oxidase NOXA regulate
sexual development in Aspergillus nidulans. Mol. Microbiol. 50,
1241–1255.
Lefebvre B, Furt F, Hartmann MA, Michaelson LV, Carde JP,
Sargueil-Boiron F, Rossignol M, Napier JA, Cullimore J,
Bessoule JJ, Mongrand S (2007) Characterization of lipid rafts
from Medicago truncatula root plasma membranes: a proteomic
study reveals the presence of a raft-associated redox system. Plant
Physiol. 144, 402–418.
Levine A, Tenhaken R, Dixon R, Lamb C (1994) H 2 O 2 from the ox-
idative burst orchestrate the plant hypersensitive disease resistance
response. Cell 79, 583–593.
Levine A, Pennell RI, Alvarez ME, Palmer R, Lamb C (1996) Calcium-
mediated apoptosis in a plant hypersensitive disease resistance
response. Curr. Biol. 6, 427–437.
Lohar DP, Haridas S, Gantt JS, VandenBosch KA (2007) A transient
decrease in reactive oxygen species in roots leads to root hair
deformation in the legume-rhizobia symbiosis. New Phytol. 173,
39–49.
Low PS, Merida JR (1996) The oxidative burst in plant defense:
Function and signal transduction. Physiol. Plant. 96, 533–542.
Margis R, Dunand C, Teixeira FK, Margis-Pinheiro M (2008) Glu-
tathione peroxidase family – an evolutionary overview. FEBS J.
275, 3959–3970.
Mehdy MC (1994) Active oxygen species in plant defense against
pathogens. Plant Physiol. 105, 467–472.
Miller G, Shulaev V, Mittler R (2008) Reactive oxygen signaling and
abiotic stress. Physiol. Plant. 133, 481–489.
Miller G, Suzuki N, Rizhsky L, Hegie A, Koussevitzky S, Mittler R
(2007) Double mutants deficient in cytosolic and thylakoid ascorbate
peroxidase reveal a complex mode of interaction between reac-
tive oxygen species, plant development, and response to abiotic
stresses. Plant Physiol. 144, 1777–1785.
ROS – Destructive or Signalling Molecules 203
Minibayeva F, Kolesnikov O, Chasov A, Beckett RP, Luthje S,
Vylegzhanina N, Buck F, Bottger M (2009) Wound-induced
apoplastic peroxidase activities: their roles in the production and
detoxification of reactive oxygen species. Plant Cell Environ. 32,
497–508.
Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004)
Reactive oxygen gene network of plants. Trends Plant Sci. 9, 490–
498.
Mongrand S, Morel J, Laroche J, Claverol S, Carde JP, Hartmann
MA, Bonneu M, Simon-Plas F, Lessire R, Bessoule JJ (2004)
Lipid rafts in higher plant cells: purification and characterization of
Triton X-100-insoluble microdomains from tobacco plasma mem-
brane. J. Biol. Chem. 279, 36277–36286.
Oldroyd GE, Downie JA (2004) Calcium, kinases and nodulation
signalling in legumes. Nat. Rev. Mol. Cell Biol. 5, 566–576.
Oldroyd GE, Downie JA (2008) Coordinating nodule morphogenesis
with rhizobial infection in legumes. Annu. Rev. Plant Biol. 59, 519–
546.
Passardi F, Penel C, Dunand C (2004) Performing the paradoxical:
how plant peroxidases modify the cell wall. Trends Plant Sci. 9,
534–540.
Passardi F, Zamocky M, Favet J, Jakopitsch C, Penel C,
Obinger C, Dunand C (2007) Phylogenetic distribution of catalase-
peroxidases: are there patches of order in chaos? Gene 397, 101–
113.
Pauly N, Pucciariello C, Mandon K, Innocenti G, Jamet A, Baudouin
E, Herouart D, Frendo P, Puppo A (2006) Reactive oxygen
and nitrogen species and glutathione: key players in the legume-
Rhizobium symbiosis. J. Exp. Bot. 57, 1769–1776.
Pei ZM, Murata Y, Benning G, Thomine S, Klusener B, Allen
GJ, Grill E, Schroeder JI (2000) Calcium channels activated by
hydrogen peroxide mediate abscisic acid signalling in guard cells.
Nature 406, 731–734.
Peleg-Grossman S, Volpin H, Levine A (2007) Root hair curling
and Rhizobium infection in Medicago truncatula are mediated by
phosphatidylinositide-regulated endocytosis and reactive oxygen
species. J. Exp. Bot. 58, 1637–1649.
Ramu SK, Peng H-M, Cook DR (2002) Nod factor induction of reactive
oxygen species production is correlated with expression of the
early nodulin gene rip1 in Medicago truncatula. Mol. Plant-Microbe
Interact. 15, 522–528.
Rouhier N, Jacquot JP (2002) Plant peroxiredoxins: alternative
hydroperoxide scavenging enzymes. Photosynth. Res. 74, 259–
268.
Rubio MC, James EK, Clemente MR, Bucciarelli B, Fedorova M,
Vance CP, Becana M (2004) Localization of superoxide dismutases
and hydrogen peroxide in legume root nodules. Mol. Plant Microbe
Interact. 17, 1294–1305.
Salzer P, Corbiere H, Boller T (1999) Hydrogen peroxide accumu-
lation in Medicago truncatula roots colonized by the arbuscular
mycorrhiza-forming fungus Glomus intraradices. Planta 208, 319–
325.
204 Journal of Integrative Plant Biology Vol. 52
Santos R, Hérouart D, Puppo A, Touati D (2000) Critical protective
role of bacterial superoxide dismutase in rhizobium-legume symbio-
sis. Mol. Microbiol. 38, 750–759.
Santos R, Hérouart D, Sigaud S, Touati D, Puppo A (2001) Oxidative
burst in alfalfa-Sinorhizobium meliloti symbiotic interaction. Mol.
Plant Microbe Interact. 14, 86–89.
Shaw SL, Long SR (2003) Nod factor inhibition of reactive oxygen
efflux in a host legume. Plant Physiol. 132, 2196–2204.
Sumimoto H (2008) Structure, regulation and evolution of NOX-family
NADPH oxidases that produce reactive oxygen species. FEBS J.
275, 3249–3277.
Takemoto D, Tanaka A, Scott B (2006) A p67Phox-like regulator is
recruited to control hyphal branching in a fungal-grass mutualistic
symbiosis. Plant Cell 18, 2807–2821.
Tanaka A, Christensen MJ, Takemoto D, Park P, Scott B (2006)
Reactive oxygen species play a role in regulating a fungus-
perennial ryegrass mutualistic interaction. Plant Cell 18, 1052–
1066.
No. 2 2010
Torres MA, Dangl JL (2005) Functions of the respiratory burst oxidase
in biotic interactions, abiotic stress and development. Curr. Opin.
Plant Biol. 8, 397–403.
Torres MA, Jones JDG, Dangl JI (2006) Reactive oxygen species
signalling in response to pathogens. Plant Physiol. 141, 373–378.
Vuletic M, Sukalovic VH, Vucinic Z (2005) The coexistence of
the oxidative and reductive systems in roots: the role of plasma
membranes. Ann. N. Y. Acad. Sci. 1048, 244–258.
Wisniewski JP, Rathbun EA, KNOX JP, Brewin NJ (2000) In-
volvement of diamine oxidase and peroxidase in insolubilization
of the extracellular matrix: implications for pea nodule initiation by
Rhizobium leguminosarum. Mol. Plant Microbe Interact. 13, 413–
420.
Wojtaszek P (1997) Oxidative burst: an early plant response to
pathogen infection. Biochem. J. 322, 681–692.
(Co-Editor: Kurt V. Fagerstedt)