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Plant Physiology 132:2196-2204 (2003) © 2003 American Society of Plant Biologists Nod Factor Inhibition of Reactive Oxygen Efflux in a Host Legume1Department of Biological Sciences, Stanford University, Stanford, California 94305–5020
Hydrogen peroxide (H2O2) efflux was measured from Medicago truncatula root segments exposed to purified Nod factor and to poly-GalUA (PGA) heptamers. Nod factor, at concentrations > 100 pM, reduced H2O2 efflux rates to 60% of baseline levels beginning 20 to 30 min after exposure, whereas the PGA elicitor, at > 75 nM, caused a rapid increase in H2O2 efflux to >200% of baseline rates. Pretreatment of plants with Nod factor alters the effect of PGA by limiting the maximum H2O2 efflux rate to 125% of that observed for untreated plants. Two Nod factor-related compounds showed no ability to modulate peroxide efflux, and tomato (Lycopersicon esculentum), a nonlegume, showed no response to 1 nM Nod factor. Seven M. truncatula mutants, lacking the ability to make nodules, were tested for Nod factor effects on H2O2 efflux. The nfp mutant was blocked for suppression of peroxide efflux, whereas the dmi1 and dmi2 mutants, previously shown to be blocked for early Nod factor responses, showed a wild-type peroxide efflux modulation. These data demonstrate that exposure to Nod factor suppresses the activity of the reactive oxygen-generating system used for plant defense responses.
The interaction of Sinorhizobium meliloti with the host legume, Medicago truncatula, results in a permissive bacterial infection and the eventual induction of a plant nodule where symbiotic nitrogen fixation occurs. The interaction is characterized by a reciprocal exchange of small molecule signals. Induction of bacterial gene expression by plant flavonoids results in the production of lipo-chitooligosaccharide signaling molecules, termed Nod factors (for review, see Dénarié et al., 1996  ; Long, 1996).
The perception of small, carbohydrate-based signaling molecules also occurs
during non-race-specific pathogen interactions (for review, see
Hahn, 1996;
Nürnberger, 1999).
Contact of the plant and microorganism leads to the liberation of plant- and
pathogen-derived oligosaccharide fragments that, in turn, trigger both
short-term changes in physiology and longer term effects through plant gene
expression (Lamb et al., 1989;
Jabs et al., 1997). Here, we
report the use of purified Nod factor and poly-GalUA (PGA) fragments to ask if
the symbiotic signal elicits an increase in oxidative radical efflux typical
for plant-pathogen interactions.
Higher plants dramatically increase the rate of reactive oxygen evolution
in response to pathogenic elicitor molecules. This oxidative burst response
cross links cell wall molecules (Bradley et
al., 1992
The bacterially derived Nod factor molecule has a small chitin backbone
with a C:16 to C:18 lipid group on the nonreducing end
(Spaink et al., 1991
Induction of both a calcium flux response and cellular growth defects in
root hair cells suggested the possibility that Nod factor might be triggering
a modified pathogenesis response. We have devised an assay to measure the
hydrogen peroxide (H2O2) efflux from excised root
segments. Using this assay on wild-type and mutant M. truncatula
plants, we demonstrate that Nod factor exposure slows the rate of
H2O2 efflux during the first hours of treatment. We
further show that the H2O2 retardation response is
specific for Nod factor and is independent or upstream of the previously
characterized calcium spiking pathway
(Catoira et al., 2000
Modulation of H2O2 Efflux in M. truncatula Root Tissue Intact root segments from M. truncatula seedlings were measured for changes in H2O2 efflux in response to purified Nod factor and a polysaccharide elicitor. The basal 1 cm of root tissue was excised from 36-h seedlings and pre-incubated for 1 h in buffered nodulation medium (BNM). Individual root segments placed in separate wells of a 96-well microtiter plate were assayed at 5-min intervals for 90 min in a fluorescence plate reader. Conversion of nonfluorescent substrate (Amplex Red) to fluorescent product (resorufin) through reaction with plant-derived H2O2 and endogenous plant peroxidases was measured using a standardized (sub-saturating) concentration of substrate (3.3 µM) and reported as accumulated fluorescence versus time (Fig. 1) or percentage of control fluorescence at 80 min (Figs. 2 and 3). Untreated root segments produced a nearly linear increase in accumulated (baseline) fluorescence over the 90-min time course of the experiment, eventually slowing as substrate became limiting (Fig. 1A).
The addition of purified S. meliloti Nod factor (RmIV C16:2, S),
applied over a biologically active concentration range from 10 pM
to 10 nM (Shaw and Long,
2003
Oligogalacturonides elicit increases in reactive oxygen evolution from many
plant families, including legumes (Levine
et al., 1994
Our goal in constructing this assay was to examine peroxide evolution from
the legume tissue that responds to nodulating bacteria in other assays (e.g.
calcium spiking, root hair deformation, and gene expression). To test that the
root segments respond to Nod factor, root hair cells on excised root segments
were assayed for the calcium spiking response
(Ehrhardt et al., 1996 BNM was used for this assay to facilitate comparisons with other assays (e.g. nodulation tests and calcium spiking response). Several properties of the buffering conditions and the nonfluorescent substrate were tested to evaluate their possible effects on the assay. Conversion of nonfluorescent substrate to fluorescent product in the absence of plant tissue, with or without Nod factor or PGA, was <1% of plant baseline substrate conversion (data not shown). Addition of Nod factor or PGA to plants in the absence of substrate produced no change in fluorescent signal (data not shown). Plants assayed in BNM and 10 nM resorufin (fluorescent product with no Amplex Red substrate) showed a <1% change in fluorescence over 90 min in all three cases: untreated, 1 nM Nod factor, or 75 nM PGA (data not shown). Titrating buffer pH from 6.5 to 7.0 resulted in a approximately 10% reduction in total fluorescence relative to controls (Fig. 2, A and B). However, the change in fluorescence for Nod factor- or PGA-treated plants for assays at pH 7.0 was not significantly different from results obtained at pH 6.5. Similar to pH change, a comparison of assay results from trials with and without 50 nM FeSO4 in the BNM indicated that the iron in the medium had little effect on the assay results (Fig. 2C). We conclude from these experiments that the conversion of nonfluorescent substrate to fluorescent product occurs only in the presence of plant tissue and that relative changes in fluorescence observed when plants are treated with Nod factor and PGA do not occur because of plant-induced changes to medium pH. Root segments cut from germinated seedlings are expected to show an initial wounding response that may include increases in H2O2 efflux. To verify that the responses to Nod factor and PGA were not caused by or requiring of a wounding response, we tested intact seedlings at a single time point. Whole seedlings were assayed by pre-incubating the root in BNM for 1 h followed by incubation in 400 µL of (3.3 µM) Amplex Red substrate and either Nod factor (1 nM) or PGA (75 nM). After 80 min, 200 µL of assay medium was transferred to a 96-well plate and immediately measured for fluorescence (Fig. 2D). Values for intact seedlings were nearly identical to those obtained using excised root segments. The contribution of injury during assay was tested by brutalizing root segments with forceps for 5 s just before assay (Fig. 2E). Damaging the root tissue immediately before assay led to an increase in peroxide efflux but did not prevent either Nod factor or PGA from achieving respective effects. The intrinsic baseline activity and the divergent responses to Nod factor and PGA in whole (uncut) seedlings argues that wounding can contribute to total assay activity but is not responsible for the baseline output or for the results obtained with either effecter molecule. Given the 20- to 30-min lag between exposure to Nod factor and slowing of H2O2 efflux, we tested the potential role of new protein synthesis using cyclohexamide, a protein synthesis inhibitor. Root segments were pre-incubated for 2 h in 5 µM cycloheximide before transfer to plates containing the assay substrate, Nod factor or elicitor treatment, and the same level of cycloheximide. Cycloheximide treatment alone elevated the levels of H2O2 efflux for all samples tested relative to solvent controls (Fig. 2F). Nod factor showed no ability to suppress H2O2 efflux in root segments treated with cyclohexamide. Moreover, PGA (75 nM) still stimulated activity, indicating that the root segments remained competent to modulate peroxide efflux.
The specificity for Nod factor's effect on suppressing H2O2 efflux was tested using two compounds structurally related to Nod factor. Application of either 1 nM sulfated chitotetraose (S-CT4) or 1 nM Nod factor lacking the reducing end sulfate required for host specificity (NodH- factor) had no measurable effect on efflux when compared with baseline levels of untreated plants (Fig. 3A). Experiments at higher Nod factor analog concentrations were not performed due to limited quantities of the reagents. Specificity for the host legume was tested by assaying a nonlegume, tomato (Solenaceae), for H2O2 efflux in the presence of 1 nM Nod factor or 75 nM PGA (Fig. 3B). Tomato (VF-36) root segments from 4-d-old seedlings were prepared in exactly the same manner as M. truncatula root segments. The application of Nod factor resulted in no significant deviation (at P = 0.05) from baseline, whereas PGA induced a striking response (Fig. 3B).
The possibility that Nod factor treatment blocks the action of the carbohydrate elicitor was tested by pre-treating plants with Nod factor and subsequently exposing them to PGA. To better approximate the rate of H2O2 production, root segments were treated and assayed in a saturating concentration of substrate (66 µM) at 1-min time intervals. Rates were calculated as the mean change in fluorescence per minute over a 30-min time period after exposure to substrate. Root segments were either assayed immediately after treatment (NF1 and PGA1 from 5–35 min) or after a 60-min pretreatment and addition of the companion treatment (NF2 and PGA2 from 65–90 min). Rate approximations were first obtained for untreated, Nod factor-treated, and PGA-treated plants (Fig. 4). Nod factor (1 nM), during the first 30 min of incubation, showed little effect on activity, whereas plants at 60 min produced only 60% of the baseline activity of untreated plants, consistent with experiments using sub-saturating substrate levels (compare Fig. 1A at 15 and 80 min with Fig. 4, NF1 and NF2). PGA (75 nM), assayed in the first 30 min, elicits a 200% increase in peroxide efflux rate compared with untreated plants, dropping to a 140% increase in rate by 60 min after PGA addition.
Pre-incubation with 1 nM Nod factor for 60 min followed by PGA (75 nM) addition resulted in an increased rate of peroxide efflux relative to both untreated and Nod factor-pretreated plants (Fig. 4). Comparing plants treated with Nod factor and then PGA (NF2-PGA1) with untreated plants (Con), the PGA stimulates the absolute level of peroxide production to only 125% of the control value instead of >200% when PGA alone (PGA1) is applied to untreated plants. If the plants treated with Nod factor and then PGA are instead compared with plants that have seen only Nod factor (NF2), the level of PGA stimulation is then about 200%. Hence, Nod factor does not block the carbohydrate elicitor peroxide response but does markedly interfere with the ability of the elicitor to increase the absolute peroxide efflux rate.
We hypothesize that the plasma membrane NADPH oxidase complex serves as the
primary generator of oxidative radicals being assayed from the root segments.
To test this hypothesis, DPI, an inhibitor of NADPH oxidase activity, was used
to assess what fraction of assay activity could be attributed to the NADPH
oxidase complex (Fig. 4). DPI
at 10 µM lowered the baseline rate to 20% of control plants.
Addition of DPI to plants either pretreated with Nod factor for 1 h or treated
directly with PGA resulted in suppression of peroxide efflux rates to 20% of
the untreated control plants. This result suggests that the NADPH oxidase
complex is responsible for the increase in efflux observed for PGA addition
and for the decrease in efflux, seen with Nod factor exposure. The conversion
of substrate to fluorescent product is specific in this assay for
H2O2 (Mohanty et
al., 1997
Mutants defective for nodule formation (nod-) have been
characterized for gene expression, intracellular calcium responses (i.e.
calcium flux and calcium spiking), and the degree of root hair deformation in
the model legume, M. truncatula (Catoira et al.,
2000
Nod Factor Does Not Elicit an Early Oxidative Burst Response
The original goal of this work was to determine if the chitin-based Nod
factor molecule, required for the rhizobia-legume symbiosis, triggers changes
in reactive oxygen generation similar to those characterized in plant-pathogen
interactions (for review, see Lamb and
Dixon, 1997
Previous studies showing an increase in reactive oxygen species during the
rhizobia-legume interaction have focused on later time points in the
development of the symbiosis. Indications from nitroblue tetrazolium staining
of whole plants at 12 to 24 h postinoculation suggest an increase in
superoxide concentration in the root (Ramu
et al., 2002
Application of purified Nod factor to M. truncatula root segments
had the surprising effect of slowing the rate of H2O2
output to 60% of baseline levels. Suppression commenced 20 to 30 min after Nod
factor application and occurred at concentrations known to elicit gene
expression and calcium spiking (Cook et
al., 1995 Using DPI, an inhibitor of NADPH oxidase activity, H2O2 efflux rates were reduced to 20% of baseline values, suggesting that the NADPH oxidase complex is responsible for the majority of peroxide efflux measured in this assay. We suspect that the residual assay activity comes mostly from the nonspecific turnover of substrate by cell wall peroxidases, with a lesser contribution coming from cellular metabolism. This supposition is based on turnover of Amplex Red in the presence of exogenous horseradish peroxidase and several attempts to quantify substrate conversion in nonliving plant material using freeze-dried root segments (data not shown).
The NADPH oxidase complex is the likely source for the majority of reactive
oxygen species produced in response to pathogenic elicitors
(Xing et al., 1997 The 20- to 30-min lag between Nod factor exposure and the observation of the peroxide retardation response suggests a possible requirement for new protein synthesis in the peroxide modulation response. Although cycloheximide blocked the ability of Nod factor to retard H2O2 production, the drug treatment itself led to an increase in peroxide generation. PGA induced efflux to levels above that seen with cycloheximide alone, indicating that PGA does not require new protein synthesis to modulate efflux rates. Although these results provide limited evidence that new protein synthesis could be required for the retardation effect, the stimulation of peroxide production (i.e. the variable we are testing) by cycloheximide strongly limits the interpretation of these data.
Plant mutants lacking the ability to make nodules show a hierarchy of
responses to Nod factor. The relationships between existing nod-
mutants have been based on assays for calcium spiking response, cellular
growth defects, gene expression, and infection competence
(Catoira et al., 2000
Two results indicate that the peroxide response and the calcium spiking
response are independent apart from the requirement for nfp.
Treatment with 10 pM Nod factor had no effect on peroxide efflux,
even though it initiates the calcium spiking response
(Ehrhardt et al., 1996
Collectively, these data suggest that a branch appears early in the Nod
factor signal transduction pathway. Low concentrations of Nod factor (<10
pM) trigger calcium spiking but do not elicit a calcium flux
response (Shaw and Long, 2003
The best characterized role for plant peroxide generation comes from
studies of the plant defense response (for review, see
Lamb and Dixon, 1997 Pretreatment of plants with Nod factor had a significant effect on the ability of PGA to increase the rate of peroxide efflux: PGA only increased the efflux rate to 125% of baseline, in comparison with 200% for plants not pretreated with Nod factor. It must be pointed out, however, that PGA stimulated the Nod factor pretreated cells from about 60% of baseline levels to 125%, or a roughly 200% change in output rate. Thus, PGA causes a substantial change in the rate of peroxide efflux even in Nod factor-pretreated plants, but the net rate of efflux is only 25% higher than the baseline efflux from untreated plants. Future studies will address the potential effects of modulating peroxide efflux on growth, the symbiotic interaction, and triggering a defense response. In conclusion, we have demonstrated using purified Nod factor that instead of the oxidative burst, found during pathogenic interactions, reactive oxygen production actually decreases in the presence of the symbiotic signal. Further, Nod factor interferes with the pathogenic elicitor when both signals are present. Given the likelihood that plants in the soil will be attacked by pathogens while undergoing permissive infection by symbiotic bacteria, it is anticipated that some aspects of the defense response will have to be ameliorated, at least locally, for successful nodulation to occur. Slowing the plant peroxide efflux rate in the presence of 100 pM to 1 nM Nod factor could help to permit the changes in cell wall growth required for root hair curling and infection, even under the pressure of pathogenic attack. Further, reducing the amount of peroxide generation may also prevent incidental triggering of defense response genes during permissive infection.
Plant Growth
Seed from wild type and mutant Medicago truncatula (Jemalong) were
treated with 70% (v/v) ethanol/water for 40 min, rinsed twice in sterile
water, and sterilized with commercial bleach for 40 min. After rinsing in
sterile water, seed were imbibed overnight at 4°C in water and germinated
overnight in inverted petri dishes. Seedlings were placed on to 1% (w/v)
agarose containing BNM (Ehrhardt et al.,
1992
Amplex Red (Molecular Probes, Eugene, OR) at 3.3 or 66 µM in BNM was made fresh from 20 mM dimethyl sulfoxide stocks. A total of 185 µL of reagent was aliquoted into each well of a 96-well microtiter plate (Microfluor2 Black, Thermo Labsystems, Franklin, MA) before addition of treatments, bringing the final volume to 200 µL. Nodulation factor NodRmIV(C16:2, Ac, and S) was diluted from 100 nM stocks in water. PGA heptamers (kind gift of Joss Rose and Mike Hahn, Complex Carbohydrate Research Center, Athens, GA) were diluted from 1 mg mL-1 stocks in water. Sulfated chitotetraose and Nod factor lacking the reducing end sulfate group (kind gifts of Dr. Jean Dénarié, Toulouse, France) were diluted from 10 nM stocks in water. Resorufin (Molecular Probes) was diluted from 100 µM stocks in water. Cycloheximide (Calbiochem-Novabiochem, San Diego) was diluted from 5 mg mL-1 stocks in dimethyl sulfoxide to final concentrations of 5 µM and 4 mM, respectively. For assay of plant segments, the basal 1 cm of each of 32 seedling was cut, placed immediately into 5 mL of BNM, and allowed to equilibrate in the dark for >1 h. Plant segments were placed into individual wells of the microtiter plate just before reading. Segments were cut to be wider than the well diameter to prevent movement during assay. Transfer of plant material to plates took approximately 6 min, resulting in a slight broadening of the assay values. Plates were read at 5- or 1-min intervals for 90 min using a Gemini fluorescence plate reader (Molecular Dynamics, Sunnyvale, CA). The plate was shaken for 2 s before each read. Dye was excited at 544 nm and read through a 570-nm dichroic mirror and a 590-nm high-pass filter. The machine was set to medium sensitivity and took the mean of six individual reads as specified by the manufacturer.
Data were exported to Excel (Microsoft, Redmond, WA) and Matlab version 7 (The Mathworks, Waltham, MA). Each treatment (e.g. Nod factor or PGA) was given to sets of six or eight plant segments per experiment, and experiments were repeated more than two times. The trials reported refer to the number of plant segments tested and used in the analysis. For instance, two experiments could consist of 16 (two x eight plant segments) data points for the control and 16 data points for Nod factor. Data were normalized between experiments by conversion to percentage of the mean of the control set for each experiment. Error estimation was calculated from normalized values including the error estimate for the control set. Histograms of values where n > 30 appeared to be normally distributed. Student's two-tailed t test for a difference in mean values was applied using functions in Excel or Matlab.
The authors wish to thank Dr. Virginia Walbot for use of her fluorescence plate reader, David Ehrhardt for constructive discussions, and the members of the Long laboratory for comments on the manuscript. Received January 28, 2003; returned for revision April 10, 2003; accepted May 15, 2003.
1 This work was supported by the Howard Hughes Medical Institute and by the Department of Energy Biosciences Division (grant no. DE–FG03–90ER2001).  * Corresponding author; e-mail squid{at}cmgm.stanford.edu; fax 650–725–8309.
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ABSTRACT
RESULTS
75
nM (0.1 µg mL-1) rapidly increased
H2O2 efflux in a dose-dependent fashion relative to
baseline levels (
