JXB Advance Access originally published online on September 10, 2004
Journal of Experimental Botany 2004 55(408):2641-2646; doi:10.1093/jxb/erh265
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RESEARCH PAPER |
Perception of Bradyrhizobium japonicum Nod factor by soybean [Glycine max (L.) Merr.] root hairs under abiotic stress conditions
Department of Plant Science, McGill University, Macdonald Campus, 21111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9
* To whom correspondence should be addressed. Fax: +1 514 398 7897. E-mail: Donald.Smith{at}McGill.Ca
Received 21 May 2004; Accepted 24 July 2004
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Abstract |
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Suboptimal growth conditions, such as low rhizosphere temperature, high salinity, and low pH can negatively affect the rhizobia–legume symbioses, resulting in poor nodulation and lower amounts of nitrogen fixed. Early stages of the Bradyrhizobium japonicum–soybean [Glycine max (L.) Merr.] symbiosis, such as excretion of genistein (the plant-to-bacteria signal) and infection initiation can be inhibited by abiotic stresses; however, the effect on early events modulated by Nod factors (bacteria-to-plant signalling), particularly root hair deformations is unknown. Thus, the objective of this study was to evaluate the perception of Nod factor by soybean root hairs under three stress conditions: low temperature, low pH, and high salinity. Three experiments were conducted using a 1:1 ratio of Nod Bj-V (C18:1, MeFuc) and Nod Bj-V (Ac, C16:0, MeFuc). Nod factor induced four types of root hair deformation (HAD), wiggling, bulging, curling, and branching. Under optimal experimental conditions root hair response to the three levels of Nod factor tested (10–6, 10–8, and 10–10 M) was dose-dependent. The highest frequency of root hair deformations was elicited by the 10–6 M level. Root hair deformation decreased with temperature (25, 17, and 15 °C), low pH, and high salinity. Nod factor concentration did not interact with either low temperature or pH. However, salinity strongly inhibited HAD responses to increases in Nod factor concentration. Thus, the addition of higher levels of Nod factor is able to overcome the effects of low pH and temperature stress, but not salinity.
Key words: Abiotic stress, Nod factor, root hair deformation, soybean
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Successful rhizobia–legume symbioses depend on efficient plant root nodulation and subsequent N2 fixation. During the early stages of infection and nodule organogenesis, bacteria attach to the plant roots in response to attractants exuded by the roots, mainly flavonoids, which activate the bacterial NodD protein, causing transcription of the bacterial nod genes. Expression of the nod genes results in synthesis of bacteria-to-plant signal molecules, or Nod factors. These compounds are composed of tri- to penta-chitin backbones and possess an N-acyl group at the non-reducing end and a variety of substitutions along the chitin backbone. Differences among Nod factors specify compatibility between macrosymbiont and microsymbiont species (Schultze and Kondorosi, 1998
). The application of an appropriate Nod factor to soybean [Glycine max (L.) Merr.] roots triggers responses such as root hair deformation and can cause the initiation of nodule primordia or the formation of complete nodule structures (Carlson et al., 1993; Stokkermans et al., 1995). It is now known that the root hair deformation elicited by Nod factors involves signal transduction through phospholipase C and D (Hartog et al., 2001). Recently, the role of phospholipase D in root hair morphogenesis was confirmed in Arabidopsis thaliana (Ohashi et al., 2003).
Abiotic stress factors, such as salinity, low pH, and low root-zone temperature can cause poor nodulation in the presence of otherwise compatible symbionts. Early events in the symbiosis such as signal production and excretion, rhizobial attachment, root hair curling, infection thread formation, and nodule initiation, are particularly sensitive to these stresses (Tu, 1981; McKay and Djordjevic, 1993; Zhang and Smith, 1996; Hungria and Stacey, 1997; Hungria and Vargas, 2000; HM Duzan, A Souleimanov, DL Smith, unpublished data).
During the early stages of symbiosis, the first visual response induced by the Nod factor in the presence of rhizobia is root hair curling. Under optimal growth conditions, the addition of purified Nod factor to legume root systems induces diverse types of root hair deformation, including, wiggling, curling, bulging, and branching (Heidstra et al., 1994; Cullimore et al., 2001; Kelly and Irving, 2002). However, how this phenomenon is affected by abiotic stress factors is not known. Thus, the biological activity of an equivalent ratio of Nod Bj-V (C18:1, MeFuc) and Nod Bj-V (Ac, C16:0, MeFuc) extracted and purified from B. japonicum and added to soybean [Glycine max (L.) Merr.] roots under three abiotic stress conditions: salinity, low pH, and low temperature was evaluated.
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Materials and methods |
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Bacterial culture
Bradyrhizobium japonicum 532C was obtained from Liphatech Inc. (Milwalkee, WI, USA). This strain was selected as it is widely used in commercial B. japonicum inoculants in Canada and the US. The culture was grown at 28 °C in 250 ml yeast mannitol broth (YEM) with shaking at 150 rpm for 4–6 d on an incubator orbital shaker (model 4580, refrigerated console, Forma Scientific Inc. USA), and thereafter subcultured into 2.0 l of YEM medium. After 7 d of subculture (OD620 0.4–0.6), bacterial cultures were induced for Nod factor synthesis and production through the addition of 5 µM genistein and incubated for an additional 48–96 h.
Nod factor extraction and purification
The resulting 2.0 l of culture was extracted with 40% HPLC-grade 1-butanol by shaking the mixture for 5–10 min and then allowing the two phases to separate for 24 h. The organic phase (butanol layer) was collected and evaporated at 80 °C in a Yamato RE500 Rotary evaporator (Yamato Scientific American Inc., NY, USA). The final volume, 2–3 ml, was dissolved in 4 ml of 18% of acetonitrile and stored in the dark in glass tubes, at 4 °C for 24 h. Samples were centrifuged for 10 min at 12 000 g, and the supernatant was collected for HPLC analysis.
For analytical purposes, 200 µl of the Nod factor extract were injected into a Waters HPLC system (Waters Associates Inc., Milford, MA) consisting of a model 712 WISP, two model 510 pumps, and a model 441 UV detector operating at 214 nm. Separation was carried out with a Vydac C18 reversed-phase column (5 µm, 46x250 mm, Vydac, USA). To elute Nod factor from the column, a programme of acetonitrile and water gradients was used: 18% acetonitrile (10 min), a linear gradient from 18% to 60% acetonitrile (20 min), and a linear gradient from 60% to 100% acetonitrile (5 min). A chromatographic peak with a retention time of 30.71 min was identified as Nod Bj-V (C18:1, MeFuc) by comparing its retention time with a standard of this Nod factor (kindly provided by Professor G Stacey, Center for Legumes Research, University of Tennessee, Knoxville, USA). A second peak with a retention time of 32.56 min was also purified by preparative HPLC. Mass spectrometry analysis (electrospray in negative mode) carried out at the Biomedical Mass Spectrometry Unit, McGill University (Dr O Mamer) allowed this peak to be identified as Nod Bj-V (Ac, C16:0, MeFuc) (Fig. 1).
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Root hair deformation assay
Soybean (cv. OAC Bayfield) seeds were surface-sterilized in 2% sodium hypochlorite for 2 min and washed thoroughly, several times, with distilled sterilized water. Two seeds were placed on each Petri plate, containing 10–20 ml 1.5% water agar, and incubated in the dark at room temperature. After 5–7 d, lateral roots of similar length and showing abundant root hairs were selected for uniformity and aseptically excised and placed on slides containing equivalent concentrations of Nod Bj-V (C18:1, MeFuc) and Nod Bj-V (Ac, C16:0, MeFuc). The slides were kept in a closed moist chamber for 24 h. Roots were fixed with a staining solution (Prithiviraj et al., 2000
). Root hair deformations were observed using light microscopy (Jenalumar, Jena Instruments Ltd, Germany) with observations focused on the root hair susceptible zone (Stokkermans et al., 1995). Each experimental unit had at least four lateral roots, on the same slide. Each treatment had three replicates. For root hair counting, the microscope field consisted of the entire length of the root hair zone of each root. The counts from all fields were added together for the determination of the percentage of root hairs deformed. The same microscopic observations were also made for the control treatment. Roots showing substantial frequencies of overlapping root hairs were not used for data collection as this overlapping tended to cause root hair deformation by itself; root hair deformations caused by Nod factor were considered only in root hairs not entangled by others (Truchet et al., 1985). With the exceptions just noted, all of the root hairs in each microscopic field were counted.
Soybean root hair response to Nod factor treatment under salinity, low pH, and low incubation temperature stress conditions
Three studies were conducted to evaluate how soybean root hairs respond to exogenous Nod factor applications under three abiotic stress factors: salinity, low pH, and low incubation temperatures. In each experiment, the treatments consisted of factorial combinations of a control where only distilled sterilized H2O was used, and three Nod factor concentrations (10–6, 10–8, 10–10 M) where the Nod factor consisted of a 1:1 mixture of Bj-V (C18:1, MeFuc) and Nod Bj-V (Ac, C16:0, MeFuc). The LCO (lipo-chito-oligosaccharide) material was freeze-dried and dissolved in distilled sterilized water to produce the required LCO solutions. Modifications were applied, based on the type of stress to be tested, as described in the following paragraphs.
To test the effect of low incubation temperature on Nod factor bioactivity, a study was carried out with three incubation temperatures (15, 17, and 25 °C). The range of incubation temperatures was chosen based on the finding of Zhang and Smith (1994), where a progressive delay of 1–2 d °C–1 in the early stages of the soybean infection process was reported as temperature declined from 25 °C (optimum) to 17.5 °C root-zone temperature (RZT). Between 17.5 °C and 15 °C the delay was much larger, at 7–10 d °C–1. Solutions of prechilled Nod factor (10–6, 10–8, 10–10, and 0.0 M) were prepared as previously described. In the control treatment, distilled sterilized water was applied to the lateral roots.
To test the effect of low pH on Nod factor bioactivity, Nod factor solutions (10–6, 10–8, 10–10 M) were adjusted to two levels of pH (4 and 5) using HCl. Two control treatments were used in this study, Nod factor solution with a control pH of 6 and, as in the first study, a distilled sterilized water control.
To test the effect of salinity on Nod factor bioactivity, two concentrations of NaCl (100 and 200 mM) were prepared by dissolving the NaCl in distilled sterilized water and mixing this with Nod factor solutions; the final volume was adjusted according to the assigned concentrations. Two controls were applied to the lateral roots: distilled sterilized water, NaCl-free Nod factor solutions.
In the three studies, soybean lateral roots of similar lengths and showing abundant fluffy root hair growth were aseptically excised and placed on microscope slides containing Nod factor solutions at a pH of 6 for salinity and low temperatures studies. To ensure complete coverage of root hairs, extra Nod factor solution was added to each specimen which was then incubated in the dark in a moist chamber at 25 °C for salinity and low pH stress factors, while for low temperature stress experiments, slides were incubated at three different temperatures (15, 17, and 25 °C). After 24 h, root hairs were stained with methylene blue (0.01%) and then scored for HAD under the microscope (Prithiviraj et al., 2000).
Experimental design and statistical analysis
The experiments were each conducted twice. Because similar data were observed in both experiments, data were combined and subjected to statistical analysis. In each experiment, the treatments were the result of factorial combinations of two factors, and the treatments were organized following a randomized split-plot design with three replicates per treatment. The main plot units consisted of the temperature, salinity, or pH levels, while Nod factor concentrations formed the subplots. The percentage deformation data for Nod factor-treated roots were square root transformed prior to statistical analysis (Steel and Torrie, 1980). The control data were excluded from the statistical analysis because root hair deformation was not detectable in this treatment. The Statistical Analysis System (SAS Institute Inc., 1990) was used for analysis of variance. When a significant treatment effect (P
0.05) was observed by ANOVA, a least significant difference (LSD) test was conducted to determine differences among means at P0.05.
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Results |
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Soybean root hair responses to Nod factor
When Nod factor was applied to soybean root hairs, four types of soybean root hair deformation were observed in this study: wiggling, bulging, curling, and branching (Fig. 2). None of the typical Nod factor-induced HADs were seen in the control treatment slides. In general, soybean root hair deformation responded in a concentration-dependent manner. Nod factor treatment increased root hair deformation, relative to the controls, in all experiments.
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Biological activity of Nod factor at low incubation temperature
The frequency of root hair deformation was dose-dependent. In general, 10–6 M Nod factor provoked the highest response (28.5%) of root hair deformations over a range of tested Nod factor. As the Nod factor concentration declined, marked reductions in HAD were observed, where 20.6% and 14.5% were the lowest average values of root hair deformation observed for Nod factor treatments at 10–8 and 10–10 M, respectively (Fig. 3). There was no interaction between Nod factor and incubation temperature. Suboptimal incubation temperature markedly affected the response of soybean root hairs to Nod factor. As the temperature decreased, lower frequencies of root hair deformation were detected at 17 °C, HAD was 82% of the control (plant roots incubated at 25 °C), while at 15 °C this was only 64% (Fig. 3).
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Biological activity of Nod factor under low pH conditions
Similar to the findings with low temperature stress, in these experiments there was no interaction between Nod factor and pH. Over all, 51% of soybean root hairs were deformed when treated with 10–6 M Nod factor at pH 6. Root hair deformation decreased as Nod factor concentration decreased; averages of 34% and 19.7% of root hairs were deformed by 10–8 and 10–10 M Nod factor, respectively (Fig. 4). The smallest response to Nod factor solution occurred at pH 4, with 29.8% of root hairs deformed. The degree of response at pH 5 was 37.8% HAD (Fig. 4).
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Biological activity of Nod factor under salinity stress
There was an interaction between salinity and Nod factor treatment. All Nod factor treatments elicited root hair deformation for the control treatment; where the proportional HAD was 42, 27, and 14% at 10–6, 10–8, 10–10 M, respectively (Fig. 5). The biological activities of NaCl-amended Nod factor solutions were markedly diminished at the two salt concentrations tested (200 and 100 mM) and there was no difference between them.
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Discussion |
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In this study the biological activity of a 1:1 mixture of two Nod factors from B. japonicum 532C was tested. Previous studies showed that the greatest number of nodules were formed when a mixture of two Nod factors (around 60:40) was applied to alfalfa root systems (Truchet et al., 1991
). Further, enod2 expression in soybean roots, an early event involved in nodule initiation, was detected only when a mixture of Nod Bj-V (C18:1, MeFuc) and Nod Bj-V (C16:0, MeFuc) was used, and their combined effectiveness was ratio-dependent, requiring an equal or higher proportion of Nod Bj-V (C18:1, MeFuc), the main Nod factor produced by microsymbionts of soybean (Minami et al., 1996).
An attempt was made to mimic stress effects, in vivo, on early stages of the perception of Nod factor in the soybean–rhizobia association (Lhuissier et al., 2001). Because of the size of soybean seeds and the nature and number of treatments required, the method described by Prithiviraj et al. (2000) was followed, which has been proven to trigger a range of types and appreciable frequencies of soybean root hair deformation when compared with other methods (Heidstra et al., 1994; Stokkermans et al., 1995) and the data from the controls showed that good frequencies of HAD were observed with this method. These data are in agreement with previous studies with respect to the frequencies of root hair deformation evoked by a variety of Nod factors and leguminous plants.
The impact of suboptimal root-zone temperatures on soybean–bradyrhizobia associations, particularly at early stages of symbiosis, has been addressed in a number of studies (Zhang and Smith, 1994, 1995, 1996; Zhang et al., 1996); evidence is presented here of the negative effects of low temperature on the bioactivity of Nod factor. Previously, the negative effect of low growth temperature on Nod factor production by B. japonicum 532C and USDA110 was addressed, a prior step in the symbiotic recognition process (HM Duzan, A Souleimanov, DL Smith, unpublished data). These data corroborate the low temperature inhibitory effect on the early stages of nodulation, specifically, on root hair curling, the first visible step in nodulation, and the step immediately following Nod signal excretion by rhizobia in the presence of a compatible legume partner (Zhang and Smith, 1994).
There was a reduction in Nod factor activity under low pH conditions. This is in agreement with previous observations regarding the low pH-sensitivity of the early steps in the nodulation process including root hair deformation, nod gene expression, Nod factor production, and root hair curling, all of which are probably related to effects on both rhizobia and the plant root system (Evans et al., 1980; Richardson et al., 1988; McKay and Djordjevic, 1993; Staley, 2003). In white clover, morphological parameters of root growth and development, such as total length, diameter, tip number, and surface area were not affected by low soil pH, while rhizobia proliferation and function were, suggesting that the inhibitory effect of low pH occurs at the microbial level, affecting nodule primordia initiation (Staley, 2002, 2003). There may be other, less direct, environmental effects on nodulation due to low pH, such as the limitation of the amount of Nod factor excreted by the microsymbiont through the reduced availability of phosphate (McKay and Djordjevic, 1993).
Salt stress decreased the soybean root hair responses to Nod factor. This is reasonable as salinity can directly restrict legume root growth, affecting the responses of roots to rhizobia and Nod factor (Tu, 1981). Salinity levels of 255 mM and 340 mM NaCl caused shrinkage of soybean root hairs, affecting the early stages of symbiosis, particularly root hair deformation (curling), resulting in an inhibition of the infection process (Tu, 1981; Elsheikh, 1998). Minor effects of osmolarity have been reported on nodD and nodABC expression in B. japonicum (Wang and Stacey, 1990).
In conclusion, as far as the authors are aware, this is the first study addressing the external response of soybean root hairs to Nod factor application under abiotic stress conditions. Under controlled environment conditions, and in the presence of low pH and low temperature stresses, the degree of root hair deformation increased with increasing Nod factor concentration, and diminished as the applied stress increased. For low temperature and pH stresses there was no interaction between Nod factor level and stress level. However, for salinity the increase in root hair deformation with increasing Nod factor concentration occurred in the control, but not when salt stress was present. Thus, adding additional Nod factor helped overcome the measured stress effect associated with low temperature and low pH, but this was not the case for salinity. Further studies would be instructive to address the effect of these stresses on Nod factor structure.
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Footnotes |
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Abbreviations: HAD, root hair deformation; LCO, lipo-chito-oligosaccharide.
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References |
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