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First published online September 23, 2005; 10.1104/pp.105.061705 Plant Physiology 139:1051-1064 (2005) © 2005 American Society of Plant Biologists
Nod Factors Induce Nod Factor Cleaving Enzymes in Pea Roots. Genetic and Pharmacological Approaches Indicate Different Activation Mechanisms1All-Russia Research Institute for Agricultural Microbiology, 196608 St. Petersburg, Pushkin, Russia (A.O.O., E.A.D., V.E.T., A.Y.B., I.A.T.); St. Petersburg State University, 199034 St. Petersburg, Russia (A.S.K.); and State Key Laboratory of Biocontrol, School of Life Sciences, SunYat-Sen (Zhongshan) University, Guangzhou 510275, China (C.S.)
Establishment of symbiosis between legumes and rhizobia requires bacterial Nod factors (NFs). The concentration of these lipochitooligosaccharides in the rhizosphere is influenced by plant enzymes. NFs induce on pea (Pisum sativum) a particular extracellular NF hydrolase that releases lipodisaccharides from NFs from Sinorhizobium meliloti. Here, we investigated the ability of non-nodulating pea mutants to respond to NodRlv factors (NFs from Rhizobium leguminosarum bv viciae) with enhanced NF hydrolase activity. Mutants defective in the symbiotic genes sym10, sym8, sym19, and sym9/sym30 did not exhibit any stimulation of the NF hydrolase, indicating that the enzyme is induced via an NF signal transduction pathway that includes calcium spiking (transient increases in intracellular Ca2+ levels). Interestingly, the NF hydrolase activity in these sym mutants was even lower than in wild-type peas, which were not pretreated with NodRlv factors. Activation of the NF hydrolase in wild-type plants was a specific response to NodRlv factors. The induction of the NF hydrolase was blocked by  -amanitin, cycloheximide, tunicamycin, EGTA, U73122, and calyculin A. Inhibitory effects, albeit weaker, were also found for brefeldin A, BHQ and ethephon. In addition to this NF hydrolase, NFs and stress-related signals (ethylene and salicylic acid) stimulated a pea chitinase that released lipotrisaccharides from pentameric NFs from S. meliloti. NodRlv factors failed to stimulate the chitinase in mutants defective in the sym10 and sym8 genes, whereas other mutants (e.g. mutated in the sym19 gene) retained their ability to increase the chitinase activity. These findings indicate that calcium spiking is not implicated in stimulation of the chitinase. We suggest that downstream of Sym8, a stress-related signal transduction pathway branches off from the NF signal transduction pathway.
Establishment of symbiosis between legumes and nitrogen-fixing rhizobia results in the formation of a new plant organ, the root nodule. Rhizobia enter the host plant usually through infection of root hairs. Bacteria within root hairs induce the formation of an infection thread that grows toward the dividing cortical cells. In a later symbiotic stage, rhizobia are released from branched infection threads into the developing nodule tissue and differentiate into nitrogen-fixing bacteroids. Nodule formation is controlled by perception of rhizobial nodulation signals: the Nod factors (NFs). Flavonoids from the host plant, in conjugation with the rhizobial activator protein NodD, induce rhizobial nodulation genes (nod, nol, and noe) that are required for NF synthesis. NFs are modified lipochitooligosaccharides, i.e. chitin oligomers linked with a fatty acid replacing the N-acetyl group on their nonreducing end (Perret et al., 2000  ; Ovtsyna and Staehelin, 2003). On host plants, NFs induce various responses at picomolar to nanomolar concentrations. These rapid changes include root hair curling (Hac; e.g. Heidstra et al., 1994), depolarization of plasma membranes (Ehrhardt et al., 1992; Felle et al., 1995), changes in H+ and Ca2+ fluxes (e.g. Felle et al., 2000), and regular oscillation of intracellular Ca2+ concentrations, called calcium spiking (Ehrhardt et al., 1996; Walker et al., 2000; Engstrom et al., 2002). When applied to roots, NFs rapidly alter the expression of various genes of the host plant and induce symbiosis-specific early nodulin (enod) genes (e.g. Albrecht et al., 1998; Pingret et al., 1998; Charron et al., 2004; Mitra et al., 2004).
Using well-characterized plant mutants unable to form nodules (Nod– phenotype), a number of plant genes controlling early symbiotic stages have been identified. These genes are part of a genetic network that controls perception and signal transduction of NFs. NF recognition seems to be mediated by the recently discovered NF receptors, i.e. Ser/Thr kinases with extracellular LysM domains (Limpens et al., 2003
NFs are active at picomolar to nanomolar concentrations, and the actual concentration of these signals in the rhizosphere is influenced by hydrolytic enzymes of the host plant. Applied to roots, purified cleavage products were nearly inactive in inducing NF responses (Heidstra et al., 1994 ; Staehelin et al., 1994). Studies with alfalfa and pea plants indicated that NFs stimulate their own degradation. Roots pretreated with nanomolar concentrations of NFs exhibited an increased NF cleaving activity in the rhizosphere (Staehelin et al., 1995; Ovtsyna et al., 2000). The NF-inducible glycosyl hydrolase released lipodisaccharides from NFs from Sinorhizobium meliloti (NodSm factors; Staehelin et al., 1994, 1995; Ovtsyna et al., 2000). The partially purified enzyme from alfalfa roots did not exhibit activity toward the polymer chitin, but cleaved the O-acetylated tetrameric NodSm-IV(C16:2, Ac, S) (Staehelin et al., 1995, 1997). The substrate and cleavage specificity of this novel NF hydrolase is therefore different from known plant chitinases that cannot release the lipodisaccharide from NodSm-IV(C16:2, Ac, S) (Staehelin et al., 1994; Minic et al., 1998; Schultze et al., 1998). Most chitinases are stress-induced enzymes involved in plant defense reactions, but certain isoenzymes appear to have a specific function in mutualistic symbioses. In the context of nodule symbioses, chitinase isoenzymes have been described to be induced at various stages of symbiosis in compatible and incompatible interactions (e.g. Staehelin et al., 1992; Vasse et al., 1993; Goormachtig et al., 1998; Xie et al., 1999; Salzer et al., 2004). The work of this article was initiated in order to understand how NFs stimulate their own degradation. Using a combination of genetic and pharmacological approaches, we found that the lipodisaccharide-forming NF hydrolase is specifically activated via an NF signal transduction pathway that includes calcium spiking. In the course of these studies, we identified a chitinase isoenzyme, whose activity was enhanced after inoculation with rhizobia and application of purified NFs, as well as various stress conditions. NFs stimulated the chitinase activity in pea mutants that lack the calcium spiking response. These findings indicate that the NF signal transduction pathway may be linked with a stress-related signal transduction pathway.
NF Degradation in the Rhizosphere and in Vitro
We have shown in a previous study that nanomolar concentrations of NodRlv factors enhance an NF cleaving activity in the rhizosphere of pea plants. NodSm factors did not stimulate the hydrolytic activity and therefore served as an inert substrate for the enzyme assay (Ovtsyna et al., 2000
The performed enzyme assay with roots from intact pea seedlings reflects the extracellular activity of the NF hydrolase. To measure the plant's total NF hydrolase activity in vitro, soluble proteins from root extracts were used for the hydrolytic assay with NodSm-IV(C16:2, S). Compared to a mock treatment with Jensen medium, plants challenged with NodRlv factors exhibited a strong lipodisaccharide-forming degradation activity in their root extracts (Fig. 1, chromatograms C and D). A similar induction of NodSm-II(C16:2) formation was also observed with the pentameric substrate NodSm-V(C16:2, S) (data not shown). These results indicate that the activity of the lipodisaccharide-forming NF hydrolase can also be measured in vitro.
As only low activity was found in noninduced roots, NodRlv factors seem to activate synthesis of the NF hydrolase. To substantiate this possibility, a number of inhibitors were tested for their capacity to reduce the NF hydrolase activity. Studies were performed with intact plants, which were concomitantly treated with NodRlv factors and the indicated inhibitors (Table II). Roots were subsequently assayed for their extracellular activity to release lipodisaccharides from NodSm-V(C16:2, S). Addition of the transcription inhibitor
To determine the minimal time required for activation of the NF hydrolase, we pretreated pea plants with NodRlv factors for different times and then determined the extracellular NF hydrolase activity with NodSm factors. Stimulation of the enzyme activity already started after 2 h, and maximal induction was reached after treatment with NodRlv factors for 20 h. Longer treatments with NodRlv factors did not increase the hydrolytic activity (data not shown).
Activation of the NF hydrolase was also tested upon incubation with rhizobial cultures. As shown previously, a pretreatment with R. leguminosarum bv viciae CIAM 1026 induced the enzyme activity. Strains producing lower levels of NFs (e.g. strain TOM) were less active in enhancing the NF hydrolase (Ovtsyna et al., 2000
Non-nodulating pea mutants of cultivars Frisson, Finale, and Sparkle (Table I) were used to characterize signaling events required for stimulation of the NF hydrolase. Plants were pretreated with 10–6 M NodRlv factors overnight and the extracellular NF hydrolase activity assayed with the substrate NodSm-V(C16:2, S). As shown in Figure 2 (chromatograms A and B), a pretreatment with NodRlv factors stimulated the NodSm-II(C16:2) forming activity in wild-type plants. In the absence of NodRlv factors, plants from cv Sparkle exhibited a relatively high constitutive activity compared to cv Finale (Fig. 3). The NF activity of all three cultivars reached similar levels when plants were treated with NodRlv factors. All nodulation mutants with a Hac– phenotype were unable to induce the NF hydrolase. When challenged with NodRlv factors, plants carrying mutations in the genes sym10, sym8, sym19, or sym9/sym30 did not show any activation of the NF hydrolase (Fig. 2, chromatograms C–E, and Fig. 3). Interestingly, these mutants produced even lower amounts of NodSm-II(C16:2) than mock-treated wild-type plants (Fig. 3). These data give genetic evidence that induction of the NF hydrolase requires a functional Nod signal transduction pathway.
Next, we extended our study on non-nodulating mutants, which are blocked at later symbiotic stages. The mutant E69 (sym7) has a Hac+ phenotype, and infection is blocked at the stage of bacterial colonization of curled root hairs. Induction experiments with this mutant showed that the NF hydrolase is enhanced to an extent comparable with that of wild-type plants (Fig. 3C). Thus, activation of the enzyme seems to be associated with rhizobial entry into root hairs.
Other mutants with a Hac+ phenotype and blocked at different stages of infection thread formation responded to NodRlv factors with increased activity of the NF hydrolase (mutants defective in the genes sym34 and sym36; Fig. 3A). NodRlv factors also stimulated the NF hydrolase activity in the mutant E135N (sym14) and in RisNod8 (sym35) with mutated nin gene (Borisov et al., 2003 Finally, we tested the induction of the NF hydrolase in hypernodulating mutants that are affected in autoregulation of nodule formation. After pretreatment with NodRlv factors, we measured increased NF hydrolase activity in the rhizosphere of the hypernodulating mutants P64 (sym28), P88 (sym29) (Fig. 3B), and RisFixC (data not shown). NF hydrolase activities in these mutants were not significantly different from those in wild-type plants. Thus, genes involved in autoregulation seem not to be required for induction of the NF hydrolase.
Using a pharmacological approach, we further studied the effect of various chemicals on the NF hydrolase activity (Table III). Calyculin A is a phosphatase inhibitor that mimics elicitor action in plant cells (Felix et al., 1994
The phytohormone ethylene inhibits the NF signal transduction pathway and blocks the NF-inducible expression of early nodulin genes in M. truncatula (Oldroyd et al., 2001 ). In our study, the ethylene releasing compound ethephon partially inhibited the activation of the NF hydrolase. Inhibition of the plant's ethylene synthesis by 2-aminoethoxyvinylglycine (AVG) did not alter the enzyme activity of the NF hydrolase. In the absence of NodRlv factors, the NF hydrolase was not induced by ethephon, the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and salicylic acid, which are known to induce pathogen-related proteins. Chitin oligomer elicitors (e.g. penta-N-acetylchitopentaose) were also completely inactive in inducing the NF hydrolase (Table III). Taken together, these data indicate that activation of the NF hydrolase is a very specific response to NFs, which cannot be mimicked by other molecules.
The extracellular NF hydrolase releases lipodisaccharides from tetrameric and pentameric NodSm factors (Ovtsyna et al., 2000
Previous studies on NF hydrolysis revealed that chitinases degrade NodSm-V(C16:2, S) to NodSm-III(C16:2) (Staehelin et al., 1994 ; Schultze et al., 1998; Ovtsyna et al., 2000). Based on these similarities, we expected that the lipotrisaccharide-forming enzyme is a chitinase that hydrolyzes glycol chitin. To visualize chitinase isoenzymes in pea roots, equal quantities of protein extracts were analyzed by electrophoresis on native polyacrylamide gels containing glycol chitin, followed by staining with a fluorescent brightener (Fig. 4, B–D). A typical chitinase isoenzyme profile from root extracts contained two bands. Leaf protein extracts and roots colonized with arbuscular mycorrhizal fungi showed additional bands in accordance with previously published data (Dumas-Gaudot et al., 1994; data not shown). The intensity of the top band varied under the conditions tested and was not further examined. Inoculation with R. leguminosarum bv viciae resulted in an increased intensity of the bottom band (marked with asterisk), indicating a Rhizobium-stimulated pea chitinase. In P56 (sym10) and RisNod10 (sym8) mutants, inoculation with R. leguminosarum bv viciae did not affect the intensity of the bottom band. All other pea mutants examined retained their capacity to increase the chitinase activity in response to R. leguminosarum bv viciae (Fig. 4, B and C). The intensity of the bottom band was also increased when NodRlv and NodSm factors were applied to the roots at micromolar concentrations (Fig. 4D). In contrast to the lipodisaccharide-forming NF hydrolase, which was activated by NodRlv factors at picomolar concentrations (Ovtsyna et al., 2000), NF concentrations <10–6 M failed to enhance the chitinase activity. Inoculation with R. leguminosarum bv viciae RBL5562, which is deficient in NF synthesis, also did not result in enhanced chitinase activity (data not shown). Taken together, these data indicate a strong correlation between the glycol chitin cleaving activity and the lipotrisaccharide-forming activity with NodSm-V(C16:2, S) as substrate. Further analysis revealed that NFs are not the only inducers of the identified chitinase. ACC (a precursor of the plant's ethylene biosynthesis), applied to roots at 10–4 M, stimulated the glycol chitin cleaving chitinase to a similar extent as NFs. An even stronger enzyme induction was seen when roots were treated with 10–3 M of salicylic acid. Moreover, a strong stimulation of the chitinase isoenzyme was observed at elevated plant growth temperature (30°C) as well as in response to excessive watering (flooding stress).
In the interaction between legumes and rhizobia, the bacterial NFs trigger multiple responses in the host plant. One of them is the rapid degradation of NFs to inactive cleavage products (Staehelin et al., 1995 ; Ovtsyna et al., 2000). In this work, we have analyzed NF cleaving pea enzymes using NodSm factors as substrates. When intact roots or protein extracts were incubated with pentameric NodSm-V(C16:2, S), two hydrolytic activities were observed. One activity released the lipodisaccharide NodSm-II(C16:2), whereas the other activity formed the lipotrisaccharide NodSm-III(C16:2). The tetrameric NodSm-IV(C16:2, S) was rapidly degraded by the lipodisaccharide-forming enzyme but was not a suitable substrate for measuring the lipotrisaccharide-forming activity. However, prolonged incubation of intact pea roots with NodSm-IV(C16:2, S) resulted in the release of low amounts of NodSm-III(C16:2) (data not shown). This is reminiscent to the lipotrisaccharide-forming activity of a chitinase lysozyme in alfalfa roots (Minic et al., 1998). The lipodisaccharide-forming pea hydrolase was specifically induced by NodRlv factors (Ovtsyna et al., 2000; this work), whereas the lipodisaccharide-forming enzyme from alfalfa was induced by NodSm factors (Staehelin et al., 1995). In pea plants, the degradation of NodSm-V(C16:2, S) to NodSm-III(C16:2) correlated with a glycol chitin cleaving activity of a chitinase isoform. The chitinase was stimulated by high doses of NFs (NodRlv factors and NodSm factors) as well as stress-related signals (ethylene and salicylic acid).
Several reports indicate that NFs induce transcriptional changes on the host plant (e.g. Charron et al., 2004
A constitutive NF hydrolase activity was measured for control plants, which were not pretreated with NodRlv factors. One possible explanation is that the NodSm factors used for the assay partially activated the enzyme in the wild-type peas. However, previous studies have shown that NodSm factors are inactive in inducing the enzyme (Ovtsyna et al., 2000 ). Interestingly, the constitutive NF hydrolase activity was significantly lower or not detectable in sym mutants that failed to induce the NF hydrolase (Fig. 3). Furthermore, a treatment with the protein phosphatase inhibitor calyculin A completely suppressed the constitutive NF hydrolase activity in the rhizosphere of wild-type plants (Table III). These data suggest that the NF signaling machinery in wild-type plants is partially active in the absence of NFs, especially in cv Sparkle. This is reminiscent of reports that certain legumes spontaneously induce nodule-like structures in the absence of rhizobia (Truchet et al., 1989; Blauenfeldt et al., 1994).
Calcium spiking (oscillation of intracellular calcium levels) is a rapid and specific response to NFs. It is suggested that calcium spiking requires extracellular Ca2+ influx as well as mobilization of Ca2+ from internal stores (Ehrhardt et al., 1996
Mounting evidence suggests that NFs induce phospholipid signaling via putative heterotrimeric G proteins (Pingret et al., 1998
The phytohormone ethylene has many effects on plant development and activates proteins related to stress and plant defense, including the chitinase studied in this work. Ethylene appears to influence nodule formation at different symbiotic stages. Exogenously applied ethylene and ethephon inhibited nodulation of pea, whereas application of the ethylene synthesis inhibitor AVG increased the number of nodules formed (e.g. Lee and LaRue, 1992
The number of nodules formed in a root system is restricted by a shoot-controlled autoregulatory mechanism of the host plant. Mutants lacking autoregulation display a hypernodulating phenotype. Sym29 and related genes in other legumes encode CLAVATA1-like receptor kinases, which are required for nodule autoregulation (Krusell et al., 2002
Chitinases stimulated during nodule symbiosis have been characterized in various host plants (e.g. Staehelin et al., 1992
Pea mutants with defective sym10 and sym8 genes failed to respond to Rhizobium inoculation with stimulation of the chitinase. In all other non-nodulating mutants examined, NFs were active in stimulating the enzyme. In other words, a set of proteins involved in activation of the NF hydrolase, namely Sym19 and Sym9/Sym30, was not required for stimulation of the chitinase. We conclude from these findings that calcium spiking and putative downstream events are not implicated in stimulation of the chitinase. We suggest that the NF perception pathway downstream of Sym8 is connected with a stress-related signal transduction pathway. In this view, chitinase stimulation would be an NF response that dissects mutants with defective sym8 and sym19 genes. Differences between these two mutants have been also reported by Novak et al. (2004) In conclusion, we show in this article that induction of an NF hydrolase in the pea rhizosphere requires a functional NF signal transduction pathway that includes calcium spiking and Sym9/Sym30 (Ca2+/calmodulin-dependent protein kinase). Stimulation of a chitinase isoform in pea roots was less dependent on the NF signal transduction pathway and only required Sym10 and Sym8. Chitinase stimulation by high doses of NFs was most likely a stress reaction. The symbiosis-related NF hydrolase inactivated excess amounts of NodSm factors in the rhizosphere, perhaps in order to attenuate NF-induced stress effects. Future work will be required to elucidate the fate of NodRlv factors in the pea rhizosphere and to identify the symbiosis-specific NF hydrolase gene.
Plant Material Seeds of pea (Pisum sativum L.) cultivars Rondo, Frisson, Finale, and Sparkle and corresponding mutants (Table I) were kindly provided by Dr. E. Jacobsen (Agricultural University, Wageningen, The Netherlands), Dr. G. Duc (Institut National de la Recherche Agronomique, Dijon, France), Dr. K.J. Engvild (Riso National Laboratory, Roskilde, Denmark), and Dr. T.A. LaRue (Cornell University, Ithaca, NY) and further propagated in St. Petersburg. Pea seeds were surface-sterilized with concentrated sulfuric acid for 5 to 10 min, washed several times with sterile water, and placed for germination on 1% water agar plates in the dark at 18°C for 3 to 4 d.
Wild-type Rhizobium leguminosarum bv viciae strain CIAM 1026 (Safronova and Novikova, 1996
Tunicamycin, the aminosteroid U73122, ethephon (2-chloroethyl-phosphonic acid), AVG, calyculin A, glycol chitosan, BHQ, and Calcofluor White M2R were obtained from Sigma-Aldrich. The other chemicals used were
NodRlv factors were purified from R. leguminosarum bv viciae strain RBL 5799 grown at 28°C on a shaker in 2 liters of B– medium (van Brussel et al., 1977
NodSm factors were purified from the overproducing strain S. meliloti 1021(pEK327). Supernatants of bacterial cultures were extracted with n-butanol, concentrated under reduced pressure, and fractionated by reverse-phase HPLC (Ultrasphere ODS, 4.6 x 250 mm or Waters, Nova Pak C18, 3.9 x 150 mm, particle size 4 µm) under isocratic conditions using 36% (v/v) acetonitrile/water containing 40 mM ammonium acetate as the mobile phase. Pentameric NodSm-V(C16:2, S) and tetrameric NodSm-IV(C16:2, S) (Schultze et al., 1992
Four or five days after sterilization, roots of germinated pea seedlings were either pretreated with a rhizobial culture (A660 = 0.05) or with NodRlv factors (10–6 M) dissolved in Jensen medium (van Brussel et al., 1982 For analysis of the NF cleaving activity in root extracts, 4-d-old seedlings were transferred into 200-mL plastic boxes containing sterile vermiculite. Inoculation with Rhizobium was performed with overnight cultures (A600 approximately 1.0). Each plant was inoculated with at least 107 to 108 bacteria. To test the effect of NFs (in Jensen medium), a first treatment (at 21°C for 24 h) was performed with seedlings placed on plastic syringes. The plants were then transferred to vermiculite and treated with a new portion of NFs. Plants were incubated in a growth chamber (at 21°C, 60% relative humidity and a light phase of 16 h with a photon flux of 400 µmol m–2 s–1). At the time of harvest, roots were washed in running water and weighed. Root tips were removed, and main roots from four to five plants from each treatment were combined and immediately frozen (–20°C).
Plant material was ground with liquid nitrogen, and the powder was suspended in 100 mM MES-NaOH buffer (pH 6.8) containing 15% ethylene glycol, 100 mM Suc, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM MgCl2. Crude homogenates were centrifuged at 14,000g for 30 min at 4°C. Contents of soluble proteins were determined by the method of Bradford (1976)
NodSm-IV(C16:2, S) or NodSm-V(C16:2, S) was used to determine the NF cleaving activity of intact roots and root extracts (Staehelin et al., 1995
The butanol phase from all samples was then dried in a speed-vac evaporator and finally dissolved in 1 µL DMSO. NodSm factors and their acylated cleavage products were fractionated on a C18 reverse-phase HPLC column at 220 nm under isocratic conditions using 35% or 36% acetonitrile/water containing 40 mM ammonium as the mobile phase. Hydrolytic rates of NFs were determined by the integration of peak areas of the substrate and the acylated cleavage products. Cleavage products were identified using the reference compounds NodSm-III(C16:2) and NodSm-II(C16:2), which have been purified and analyzed previously (Staehelin et al., 1994
Soluble protein extracts were analyzed by 12% (w/v) PAGE under native conditions according to Davis (1964)
We thank T. Artamonova (Russian Scientific Center Applied Chemistry, St. Petersburg, Russia) for mass spectroscopic analysis. We are indebted to E. Kondorosi (Institut des Sciences du Végétal, Centre National de la Recherche Scientifique) and H.P. Spaink (Leiden University) for providing bacterial strains. We express our gratitude to W.J. Broughton (University of Geneva, Switzerland) for his assistance with many aspects of this work. Received February 21, 2005; returned for revision June 14, 2005; accepted June 19, 2005.
1 This work was supported in part by the Russian Academy of Agricultural Sciences, a grant of the President of Russian Federation (HIII–1103.2003.04), by the Russian Ministry of Science and Technologies, and by a grant from the U.S. Civilian Research and Development Foundation (CRDF ST–012–0).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.061705. * Corresponding author; e-mail lsdcs{at}zsu.edu.cn; fax 862084115516.
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-glucuronidase fusion in alfalfa (Pingret et al., 1998
nodA; defective in synthesis of NFs; Wijffelman et al., 1985