JXB Advance Access originally published online on September 10, 2004
Journal of Experimental Botany 2004 55(408):2635-2640; doi:10.1093/jxb/erh261
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RESEARCH PAPER |
Nod factor-treated Medicago truncatula roots and seeds show an increased number of nodules when inoculated with a limiting population of Sinorhizobium meliloti*
1Department of Agronomy and Soils, University of Puerto Rico—Mayagüez, PO Box 9030, Mayagüez, PR 00681-9030, USA
2Laboratoire des Interactions Plantes-Microorganismes, INRA/CNRS, Chemin de Borde-Rouge, BP 27, F-31326 Castanet-Tolosan, France
Present address and to whom correspondence should be sent: Biological Sciences Department, California State Polytechnic University, 3801 W. Temple Avenue, Pomona, CA 91768, USA. E-mail: gbrelles{at}csupomona.edu
Received 12 July 2004; Accepted 21 July 2004
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Abstract |
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Medicago truncatula is a model legume plant that interacts symbiotically with Sinorhizobium meliloti, the alfalfa symbiont. This process involves a molecular dialogue between the bacterium and the plant. Legume roots exude flavonoids that induce the expression of a set of rhizobial genes, the nod genes, which are essential for nodulation and determination of the host range. In turn, nod genes control the synthesis of lipo-chito-oligosaccharides (LCOs), Nod factors, which are bacteria-to-plant signal molecules mediating recognition and nodule organogenesis. M. truncatula roots or seeds have been treated with Nod factors and hydroponically growing seedlings have been inoculated with a limiting population of S. meliloti. It has been shown that submicromolar concentrations of Nod factors increase the number of nodules per plant on M. truncatula. Compared with roots, this increase is more noticeable when seeds are treated. M. truncatula seeds are receptive to submicromolar concentrations of Nod factors, suggesting the possibility of a high affinity LCO perception system in seeds or embryos as well.
Key words: Legume nodulation, Medicago truncatula, Nod factors, Sinorhizobium meliloti
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Soil bacteria belonging to the genera Rhizobium, Bradyrhizobium, Sinorhizobium, Allorhizobium, and Mesorhizobium (collectively known as rhizobia) are able to interact with roots, and sometimes stems, of legume plants and to form specialized structures called nodules where nitrogen fixation takes place. For this process to occur there has to be a molecular dialogue between the plant and the bacterial partner. Legume (and non-legume) roots exude compounds, such as flavonoids, that induce the expression of a set of rhizobial genes, the nod genes, which are essential for nodulation and determination of the host range. nod genes are responsible for the synthesis of the rhizobial Nod factors (NFs) (reviewed in Cullimore et al., 2001
; Hirsch et al., 2001). NFs are bacteria-to-plant signal molecules that trigger responses that include the formation and deformation of root hairs, membrane depolarization, intra- and extracellular alkalinization, the induction of early nodulin gene expression, changes in ion fluxes, and the formation of nodule primordia (Broughton et al., 2000; Cullimore et al., 2001; Guerts and Bisseling, 2002; Perret et al., 2000; D'Haeze and Holsters, 2002).
The structure of NFs was first determined by Lerouge et al. (1990) for Sinorhizobium meliloti; it is now known that all rhizobia produce NFs having the same generic structure. NFs are lipo-chito-oligosaccharides (LCOs) generally composed of three to five N-acetyl glucosamines, ß-1-4-linked with the N-acetyl group of the terminal non-reducing end replaced by a fatty acid of 16–18 carbon residues. NFs can be specifically modified with acetate, sulphate or carbamoyl groups or can have attached sugars such as arabinose or fucose (Dénarié et al., 1996).
Purified NFs per se are able to initiate complete nodule structures at submicromolar concentrations (Dénarié and Cullimore, 1993). It has been reported that submicromolar concentrations of NFs induce physiological changes in legume hosts (reviewed in D'Haeze and Holsters, 2002; Goedhart et al., 2003) and non-legume plants as well (Prithiviraj et al., 2003; Souleimanov et al., 2002). At submicromolar concentrations NFs affected the germination of seeds of some crop plants (Prithiviraj et al., 2003). When using Bradyrhizbium japonicum NF, Nod Bj-V C18:MeFuc, the maximum effect was observed for the host plant, soybean, which resulted in 90% germination after 72 h of treatment, while the germination level in the control treatment was only 40% (Prithiviraj et al., 2003). It was also shown that NF, when present in a hydroponic solution, enhanced the growth of soybean plants and significantly increased the nodule dry weight without an increase in the total number of nodules (Souleimanov et al., 2002).
S. meliloti, the alfalfa symbiont, produces four major NFs: NodSm-IV (Ac,S), NodSm-IV (S), NodSm-V (Ac,S), and NodSm-V (S), with a C16:2 N-acyl chain for all factors. Other minor compounds that differed slightly from NodSm-IV (S) in the length of the aliphatic chain (C18) and in the number and location of the double bonds were detected (Roche et al., 1991b). (For nomenclature and detailed structures see Dénarié et al., 1992.) Purified NodSm-IV (Ac,S) and NodSm-IV (S) factors show biological activity on alfalfa. At concentrations down to 10–12 M they elicit root-hair deformation (Lerouge et al., 1991; Roche et al., 1991a). Cortical cell divisions and the formation of genuine nodules are elicited on alfalfa at concentrations down to 10–9 M (Truchet et al., 1991).
How plants perceive NFs is unclear. The presence of more than one NF perception system has been suggested for the last decade (Ardourel et al., 1994). Several candidates have been proposed as putative NF receptors (Cullimore et al., 2001). Two distinct NF binding sites, called NFBS1 and NFBS2, with different affinities have been characterized in Medicago spp. (Bono et al., 1995; Gressent et al., 1999). It has recently been reported that two LYK (LysM domain-containing receptor-like kinases) genes are specifically involved in infection thread formation in Medicago truncatula. This, together with the properties of the LysM domain, suggested that they are Nod factor entry receptors (Limpens et al., 2003). The cloning of a putative Nod-factor kinase gene (NFR5) from Lotus japonicus, essential for NF perception, has been recently reported as well (Madsen et al., 2003).
Medicago truncatula is a legume that establishes symbiotic interactions with S. meliloti and has been widely used as a plant model in recent years (Cook et al., 1997; Cook, 1999). As far as the authors are aware, there are no reports regarding the effect of NFs on M. truncatula nodule numbers and the question of whether seeds perceive NFs in a way that affects nodulation has not been addressed before. The aim of this work was to determine if there is an effect on the nodulation of M. truncatula inoculated plants, by treating seeds or roots with NFs.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Bacterial strain and suspension preparation
To determine the bacterial limiting population, Sinorhizobium meliloti strain 2011 was grown either in tryptone-yeast extract (TY) (Beringer, 1974
) or in Vincent minimal medium (Vincent, 1970) until the exponential growth phase. The bacterial culture was filtered through a 0.22 µm filter. The biomass was resuspended in sterile distilled water, OD was determined and suspensions were made in order to have 0, 10, 103, 105, and 107 bacteria ml–1. Each Medicago truncatula seedling, growing in plastic growth pouches, was inoculated using 0.5 ml of these suspensions. To study the effect of NFs on the nodulation of M. truncatula plants, the bacterial strain was grown in TY medium, filtered, and resuspended, as indicated above, and 0.5 ml of a 2x103 bacteria ml–1 suspension used to inoculate each plant seedling.
Nod factor source
NF experiments were carried out with a HPLC-purified wild-type S. meliloti Nod-factor mixture consisting mainly of NodSm-IV (C16:2,Ac,S) (Roche et al., 1991b). A stock solution (10–3 M in water:ethanol 1:1, v:v) was used to prepare water-diluted solutions at the appropriate concentrations.
Determination of S. meliloti limiting population
Seeds of M. truncatula cv. Jemalong A17 were surface-sterilized with H2SO4 for 7 min and rinsed six times with sterile distilled water. Seeds were transferred to Petri dishes containing soft agar and gibberellic acid (1 µM) and germinated at 25 °C in the dark for 1 d. Germinated seeds were transferred to plastic growth pouches (four seeds per pouch) containing 9 ml of Fahraeus medium (Fahraeus, 1957). Pouches were placed in a growth chamber at 25 °C, 16/8 h day/night cycle, light at 65 µM m–2 s–1.
Three to four days after transferring the seedlings, each root was inoculated with 0.5 ml of a suspension of 0, 20, 2 x103, 2x105, and 2x107 bacteria ml–1. Plants were checked daily for nodule appearance and the number of nodules was scored each day. Each treatment consisted of 10 growth pouches with four seeds per pouch. Pouches for each treatment were randomly chosen and experiments were repeated four times.
Effect of the addition of Nod factors on M. truncatula nodulation
Root treatment: Seeds of M. truncatula cv. Jemalong A17 were surface-sterilized as described above. Seeds were transferred to Petri dishes containing soft agar and gibberellic acid (1 µM) and germinated at 25 °C in the dark for 1 d. Germinated seeds were transferred to plastic growth pouches containing 9 ml of Fahraeus medium and the appropriate concentration of NFs (0, 10–7, 10–8, and 10–9 M). Pouches were placed in a growth chamber at 25 °C, 16/8 h day/night cycle, light at 65 µM m–2 s–1.
When root seedlings were approximately 5–8 cm long (2–3 d after transferring to pouches), they were inoculated with 0.5 ml of a 2x103 bacteria ml–1 S. meliloti strain 2011 suspension prepared as described above. Plants were checked daily for nodule appearance and the number of nodules was scored each day.
Each treatment consisted of 10 growth pouches with four plants per pouch. Pouches for each treatment were randomly chosen and experiments were repeated four times.
Seed treatment: Seeds of M. truncatula cv. Jemalong A17 were surface-sterilized as described above. Sterile seeds were soaked in 10 ml of a Fahraeus solution of NFs (0, 10–7, 10–8, and 10–9 M) dispensed into a Petri dish, gently shaken for 1 h and blotted dry with sterile filter paper. Seeds were transferred to Petri dishes containing soft agar and gibberellic acid (1 µM) and germinated at 25 °C in the dark for 1 d. Germinated seeds were transferred to plastic growth pouches containing 9 ml of Fahraeus medium. Pouches were placed in a growth chamber at 25 °C, 16/8 h day/night cycle, light at 65 µM m–2 s–1.
When root seedlings were approximately 5–8 cm long (2–3 d after transferring to pouches), they were inoculated with 0.5 ml of a 2x103 bacteria ml–1 S. meliloti strain 2011 suspension prepared as described above. Plants were checked daily for nodule appearance and the number of nodules was scored each day.
Each treatment consisted of 10 growth pouches with four plants per pouch. Pouches for each treatment were randomly chosen and experiments were repeated four times.
Data analysis
Data were analysed by ANOVA using the SAS System (SAS Institute Inc., 1999). Treatment means of the number of nodules per plant were compared using the GLM Procedure with the LSD test.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Preliminary experiments were carried out to determine the minimal rhizobia population size that produces consistent plant nodulation. ‘Limiting rhizobia population’ was defined as the bacterial population size below which there is no noticeable plant nodulation and, in this case, it was determined to be 103 bacteria ml–1. The bacterial culture medium that produced the most reproducible results was TY (data not shown). This limiting rhizobia population was used as a suspension to inoculate seedlings of M. truncatula that were in contact with NFs before seed germination (seed treatment) or during plant development (root treatment).
Nod factors, when present in the hydroponic solution in pouches or in the pre-soaking solutions in Petri dishes, increased the number of nodules of M. truncatula plants (Table 1). The NF concentration that gave the best result was 10–9 M for both root and seed treatment. NF-treated plants also seemed to have a better-developed root system since there were more and longer secondary roots compared with the control plants (data not shown). The first set of experiments (root treatment) gave a preliminary indication of the effect of NFs on M. truncatula nodulation (Fig. 1; Table 1). Although there was an increase in the number of nodules per plant for NF-treated roots, differences between means were not significant. For this reason, a refinement of the first protocol has been developed to determine whether treating seeds with NFs could influence nodule numbers as well. It is important to point out that although absolute values were different for root and seed experiments, there was always an increase in the number of nodules per plant after the NF treatment.
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The effect of presoaking seeds with NF solutions on M. truncatula nodulation can be seen in Figs 2 and 3. Already, at 7 d after inoculation, 10–9 M NF caused a 109% increase in the number of nodules per plant, compared with the control. However, means were not significantly different at this stage. Eleven days after inoculation, 10–9 M NF seed-treated M. truncatula plants showed a 119% increase in the number of nodules per plant compared with the control. Fifteen days after inoculation, the number of nodules per plant was 37%, 69%, and 78% higher compared with the control for 10–7 M, 10–8 M, and 10–9 M Nod factor concentrations, respectively (Table 1). There was no effect of NFs on the germination of seeds (data not shown). However, seeds were germinated in the presence of gibberellic acid. This compound accelerates seed germination and could have masked a possible effect due to the NF treatment.
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A considerable amount of work regarding the effect of NF responses and their structural requirements has been published (reviewed in D'Haeze and Holsters, 2002
). Most of that work has been carried out by treating legume root hairs with NFs, since responses are generally more intense in the region of the root that is susceptible to nodulation. There are few studies on the effect of NFs on nodule numbers. It has been reported that there is a significant increase in the nodule dry weight for NF-injected soybean plants as compared with control (water-injected) plants. However, this effect was due to an increase in the weight per nodule and not in the total number of nodules (Souleimanov et al., 2002). van Brussel et al. (2002) reported that the application of NFs from Rhizobium leguminosarum bv. viciae to one side of a split-root system of Vicia plants inhibited nodule formation, and thus nodule number, on the other side. These results indicate that NFs elicit an autoregulatory response. A similar result was reported by Catford et al. (2003) in a split-root system of alfalfa treated with NodSm-IV (C16:2, S) from S. meliloti. As far as is known, there are no reports regarding the effect of NFs on M. truncatula nodule numbers. Moreover, there are no reports regarding the effect that NF treatment of seeds might have on the formation of rhizobia-induced nodules.
For the root treatment, increases in the number of nodules per plant for treatments, compared with the control, are smaller and appear later than in the seed treatment (119% increase for seed-treated plants versus 30% increase for root-treated ones, 15 d and 20 d after inoculation, respectively, and both for 10–9 M NF). This effect is intriguing considering the constant presence of NFs in the growth pouch during plant development that might induce a continuous stimulation of the NF perception system(s) in root-treated plants. Under these conditions, NF concentration at the receptors might be high considering the concentrating effect of the cell wall (Cullimore et al., 2001). However, it has been reported that the chitin backbone of NFs can be hydrolysed by plant chitinases (reviewed in D'Haeze and Holsters, 2002). In Medicago sativa, the natural S. meliloti host, NFs induce their own breakdown by enhancing the production of a ‘dimer-forming hydrolase’ (Staehelin et al., 1995). This NF-hydrolase is induced by NodSm-IV (C16:2,Ac,S). It has been speculated that a rapid NF-induced degradation of NFs may be part of NF signalling, required for the induction of plant genes involved in nodule development, or necessary to finely regulate NF amounts and to avoid a continuous stimulation of the NF perception system(s) (Staehelin et al., 1995) preventing the elicitation of defence-like reactions (Savouré et al., 1997). More recently, M. truncatula chitinase genes that are expressed during nodule development were isolated and characterized (Salzer et al., 2000). If LCOs are hydrolysed by plant enzymes and degradation products are not recognized by the plant, a constant presence of NFs in the growth pouch would not lead to constant increases in the number of nodules per plant once the receptor(s) systems are saturated.
Catford et al. (2003) have reported a decrease of nodule numbers in split-root systems of alfalfa treated with NodSm-IV (C16:2, S) prior to S. meliloti inoculation indicating the presence of an autoregulatory feedback mechanism. In this case, they have used a higher inoculum density (2x108 bacteria ml–1) compared with the limiting rhizobial population that was used in this study. Moreover, they have made daily applications of 4 ml of a 10–8 M NF solution from 8 d prior to inoculation until harvest (22 d in total). Therefore, the total NF concentration is higher than in this study's experiments. It might be possible that a high NF concentration induces autoregulation, while a low one induces nodule number increases.
The smaller increases in nodule numbers that are reported here for root treatment, compared with seed treatment, might be due to some autoregulatory effect. The traces of NFs possibly remaining on seeds in the seed treatment (see below) might be sufficient to induce nodulation enhancement, but not autoregulation.
Seed-treated plants showed an increased nodulation compared with the control. After NF treatment, seeds were blotted dry with sterile filter paper and then transferred to soft agar for germination. Part of the absorbed NFs might diffuse into the agar medium during seed germination. As seeds are not rinsed after treatment, traces of NFs may remain during plant development, leading to increased nodulation. However, the amount remaining on seeds should be very limited. Alternatively, plants might somehow ‘remember’ the NF imbibing solution that seeds (and possibly embryos) received. Many indications support the hypothesis that NFs are perceived by plant receptors (reviewed in D'Haeze and Holsters, 2002). In Medicago spp. two different NF binding sites, with different affinities, have been described, NFBS1 and NFBS2 (Bono et al., 1995; Gressent et al., 1999). It has been recently reported that two LYK (LysM domain-containing receptor-like kinases) genes are specifically involved in infection thread formation in M. truncatula. These kinases have been suggested as Nod factor entry receptors (Limpens et al., 2003). No attempt, as far as is known, has been made to address the question of whether NFs are perceived by seeds and/or embryos as well. It is important to point out that the germination process is triggered by the surface-sterilization and sulphuric acid scarification of seeds. Thus, the NF imbibing solution is not in contact with an intact seed, but one that is already undergoing germination. Therefore, NF might be perceived by the developing embryonic root and not by putative NF receptors on the seed surface.
Are there NF receptors in seeds/embryos or do seeds ‘memorize’ the fact of having been in contact with NFs? At this time, there are no experimental results to answer these questions, but further work is being carried out to test this hypothesis.
In conclusion, increased plant nodulation is achieved by treating M. truncatula roots and mainly seeds with NFs prior to inoculation. These results might have technological applications since presoaking alfalfa seeds with NFs before sowing may lead to increased nodulation under field conditions as well. In fact, a new soybean inoculant based on NF technology has been put on the market very recently (Osburn et al., 2004).
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Acknowledgements |
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Purified Nod factors and Medicago truncatula seeds were kindly provided by Clare Gough and Jean Dénarié and Etienne P Journet, respectively. The authors gratefully acknowledge Jean Dénarié and Clare Gough for critically reviewing the manuscript and helpful suggestions. GBM wants to acknowledge Estación Experimental del Zaidín, CSIC (Granada, Spain) for the use of the library facilities. GBM is indebted to the Centre National de la Recherche Scientifique (CNRS) France, for a Research Associate position.
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Footnotes |
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* This paper is dedicated to the late Graciela Labourdette.
Abbreviations: NF/NFs, Nod factor/s; LCO, lipo-chito-oligosaccharides; TY, tryptone-yeast extract.
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References |
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