Journal of Experimental Botany, Vol. 52, No. 364, pp. 2181-2186,
November 1, 2001
© 2001 Oxford University Press
Original Papers |
Redifferentiation of bacteria isolated from Lotus japonicus root nodules colonized by Rhizobium sp. NGR234
Botanical Institute, Hebelstrasse 1, CH-4056 Basel, Switzerland
Received 30 April 2001; Accepted 20 June 2001
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Abstract |
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In most studies concerning legume root nodules, the question to what extent the nodule-borne bacteroids survive nodule senescence has not been properly addressed. At present, there is no ‘model system’ to study these aspects in detail. Such a system with Lotus japonicus and the broad host range Rhizobium sp. NGR234 has been developed. L. japonicus L. cv. Gifu was inoculated with Rhizobium sp. NGR234 and grown over a 12 week time period. The first nodules could be harvested after 3 weeks. Nodulation reached a plateau after 11 weeks with a mean of 64 nodules having a biomass of nearly 100 mg FW per plant. Nodules were harvested and homogenized at different stages of plant development. Microscopic inspection of the extracts revealed that, typically, nodules contained c. 15x109 bacteroids g-1 FW, and that about 60% of the bacteroids were viable as judged by vital staining. When aliquots of the extracts were plated on selective media, a substantial number of ‘colony-forming units’ was observed in all cases, indicating that a considerable fraction of the bacteroids had the potential to redifferentiate into growing bacteria. In nodules from the early developmental stages, the fraction of total bacteroids yielding CFUs amounted to about 20%, or one-third of the bacteroids judged to be viable after extraction, and it increased slightly when the plants started to flower. In order to see how nodule senescence affected the survival and redifferentiation potential of bacteroids, some plants were placed in the dark for 1 week. This led to typical symptoms of senescence in the nodules such as an almost complete loss of nitrogenase activity and a considerable decrease in soluble proteins. However, surprisingly, the number of total and viable bacteroids g-1 nodule FW remained virtually constant, and the fraction of total bacteroids yielding CFUs did not decrease but significantly increased up to 75% of the bacteroids judged to be viable after extraction. This result indicates that during nodule senescence bacteroids might be induced to redifferentiate into the state of free-living, growing bacteria.
Key words: Nitrogen fixation, senescence, symbiosis, vital staining.
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Introduction |
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Concerning the interaction between legumes and rhizobia, most of the research efforts have been focused on the initial phase of this interaction, namely nodule morphogenesis and the onset of nitrogen fixation (Sanchez et al., 1991
). To what extent the potential of rhizobia to grow and divide is preserved during the lifetime of a nodule and, finally, if and how they get out of nodules has been much less well examined, although the release of rhizobia from nodules is thought to be crucial for the evolution of these nitrogen-fixing bacteria (Sprent and Raven, 1992). It may occur upon stochastic degradation of nodules, for example, by soil-borne animals, or upon nodule senescence. Nodule senescence occurs in a determinate way in annual legumes after pod filling and stochastically upon environmental perturbations like defoliation, darkening, and water deficit (Vikman and Vessey, 1993a, b, c). Senescence is characterized by an increase in proteolytic activities (Pfeiffer et al., 1983; Vincent and Brewin, 2000), especially with respect to the degradation of leghemoglobin (Lb; Pladys et al., 1991; Vikman and Vessey, 1993a, b, c; Moreau et al., 1996) and an increase of reactive oxygen species (Evans et al., 1999; Matamoros et al., 1999a, b). These findings, along with EM-studies on bacteroids in senescing nodules (Pladys and Rigaud, 1988) have led to the view that bacteroids are more or less completely digested during nodule senescence (reviewed in Udvardi and Day, 1997). However, the survival of bacteria has not been adequately addressed in most studies on nodule senescence (cf. Vikman and Vessey, 1993a, b, c; Swaraj et al., 1993), and there is some experimental evidence that bacteroids could escape from lysis in senescent nodules. Bacteroid proteins seem to be protected to a large extent from overall proteolysis in senescing nodules (Pfeiffer et al., 1983; Sarath et al., 1986), perhaps due to the presence of protease inhibitors (Garbers et al., 1988; Manen et al., 1991). A more direct indication that a non-negligible part of nodule bacteroids may escape lysis and redifferentiate into growing bacteria comes from re-isolation studies. For example, in soybean nodules undergoing senescence upon nitrate treatment or upon treatment by a herbicide, the number of viable bacteria recovered from the nodules did not decrease as compared to non-senescing control nodules (Müller et al., 1994, 2001). Moreover, in an advanced stage of nodule senescence, trehalose, a bacteroid-borne disaccharide, accumulates strongly and becomes the most abundant soluble carbohydrate, indicating that bacteroids continue to be biosynthetically active and to adapt themselves while the plant tissue degenerates (Müller et al., 2001; see also Streeter, 1981). These findings contradict the view that bacteroids are completely digested, but indicate instead that they maintain an ability to redifferentiate. Currently, a ‘model system’ allowing studies of the molecular basis of this aspect of nodulation is lacking.
Lotus japonicus has well-established classical and molecular genetics, especially with respect to nodulation (Jiang and Gresshoff, 1997; Schauser et al., 1999). The nodules of L. japonicus nodules are somewhat special because they are determinate like the ones of soybean and other tropical legumes, but they export amides instead of ureides (Sprent, 1980). The broad host range Rhizobium sp. NGR234 (Pueppke and Broughton, 1999) efficiently nodulates L. japonicus (Hussain et al., 1999). The symbiotic plasmid of this strain has been completely sequenced (Freiberg et al., 1997). Thus, L. japonicus–Rhizobium sp. NGR234 could be a system of choice to study the late phase of nodulation, especially with respect to nodule senescence and the maintenance of vitality of the microsymbiont.
Here, data are presented obtained from L. japonicus nodules colonized by Rhizobium sp. NGR234 harvested over a 12 week time period and after senescence induced by darkening. Bacteroids were extracted from nodules, stained with a vital stain or plated on a selective medium in order to monitor their ability to redifferentiate into growing free-living bacteria.
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Materials and methods |
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Plant material
Lotus (L. japonicus L. cv. Gifu) seeds were scarified by immersion in concentrated H2SO4 for 10 min, washed well with tap water, surface-sterilized by immersion in 30% (v/v) H2O2, and germinated on 1% water agar. After 4 d, more than 90% of the seeds had germinated. Seedlings were then transferred to Magenta jars. The upper compartment contained 100 ml of a Perlite:Vermiculite 1:1 (v/v) mixture. The lower part was filled with 200 ml 0.25x concentrated B&D-nutrient solution (Lewin et al., 1990
). After 2 d, the jars were inoculated with 1 ml of a stationary culture of Rhizobium sp. NGR234 grown in 20E-medium (Stripf and Werner, 1978). Plants were grown in a phytotron under long day conditions (16 h day, 160 µE m-2 s-1, 22 °C; night at 18 °C; RH 70%). The plants were harvested at various time points (3 plants per time point) and the nodules were processed for further analyses. 11 weeks after inoculation, senescence was induced on six plants by darkening (less than 0.05 µE m-2 s-1) for 1 week. Darkening was chosen as a way to induce senescence in order to be compliant with the most recent studies on induced nodule senescence (Matamoros et al., 1999a, b).
Analytical
Growth was followed by determining shoot DW after lyophilization of shoots for 2 d.
Nitrogenase was assayed using entire root systems and the acetylene reduction assay (ARA) as described previously (Müller et al., 1994). ARA was established as a routine way to estimate nitrogenase activity although it is known to have errors due to nodule disturbance. The activity was calculated by subtracting the amount of ethylene formed 2 min after adding acetylene to the roots from the amount formed 30 min later and expressed in pmol acetylene reduced s-1 g-1 nodule FW (pkat g-1 FW).
After measuring nitrogenase, nodules were harvested, weighed, surface-sterilized by 10 min immersion in H2O2 (30% v/v), subsequently washed four times in 10 vols 0.2 M mannitol buffered with 20 mM phosphate/K+ (pH 7.5) and homogenized in 5 vols of the same buffer using a plastic pistil. To determine soluble protein contents, aliquots of the crude extracts were centrifuged in order to pellet bacteroids and cell debris (10000 g at 4 °C, 10 min). To determine total protein contents, aliquots of the crude extracts were supplemented with Triton-X-100 (1% final concentration), freeze–thawed once and thoroughly vortexed. Protein was determined in the supernatants or in the crude homogenates after convenient dilution using microtiter plates (Bradford, 1978).
In order to quantify bacteroids and their potential to redifferentiate into growing free-living bacteria, a dilution series of the crude extracts was made. To count living and dead bacteroids immediately after extraction, aliquots of convenient dilution steps were stained according to the manufacturer's instructions by the Live/Dead® BacLightTM bacterial viability kit (Molecular Probes, Eugene, Oregon, USA) containing two nucleic acid stains, SYTO® 9 and propidium iodide. Stained suspensions were mounted in a Thoma counting chamber and excited at 470 nm under a fluorescence microscope (Axioplan, Zeiss, Jena, Germany). Viable and non-viable bacteroids were counted as green (maximum emission wavelength of SYTO® 9 at 530 nm) and red (maximum emission wavelength of propidium iodide at 620 nm), respectively. The sum of both viable and non-viable bacteroids is referred to as the total number of bacteroids. To evaluate the potential of bacteroids to redifferentiate into growing, free-living bacteria, aliquots of convenient dilution steps were plated on 20E-medium amended with 0.2 M mannitol as described (Müller et al., 1994). The medium contained rifampicine (50 mg l-1) and cycloheximide (100 mg l-1). Plates were incubated at 27 °C and colonies were counted after 8 d. Each colony-forming unit (CFU) was taken to correspond to a bacteroid that had redifferentiated into a growing and dividing bacterium.
Statistics
Analyses of variance and Student–Newman–Keuls tests were performed using the software SigmaStat (Jandel Scientific, San Rafael, CA, USA).
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Results |
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Kinetics of growth, nodulation, nodule protein contents
L. japonicus seedlings were inoculated with Rhizobium sp. NGR234, grown in a phytotron and harvested between 3 and 12 weeks post-inoculation (wpi), thus between 4 and 13 weeks after sowing. Shoot DW remained more or less constant until 6 wpi and then started to increase exponentially (Fig. 1
). This increase was preceded by an apparent sharp increase of nitrogenase activity between 5 and 6 wpi (Fig. 1). The first plants started to flower after 8 weeks.
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Nodule FW was lower than 1 mg per plant until 4 wpi and then exhibited an initial increase to reach 7 mg per plant at 5 wpi (Fig. 2
). At 11 wpi, nodule FW reached nearly 100 mg per plant. Between 3 and 9 wpi, the number of nodules per plant increased by a factor of 10, namely from a mean number of 6 to 64 nodules per plant (Fig. 2).
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Because of the low nodule FW at the beginning of the harvesting period, nodule extracts could only be made from 4 wpi onwards. On a fresh weight basis, total protein contents of the nodules increased until 7 wpi with a maximum at 13 mg g-1 FW and declined thereafter. The changes of soluble and total protein contents had a very similar profile (Fig. 3
).
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Induction of nodule senescence
After 11 wpi, when nodulation reached a relative maximum, six flowering plants were subjected to artificial senescence by darkening for 1 week (cf. Matamoros et al., 1999a, b). The senescent plants were compared to non-senescent, flowering plants and, furthermore, to non-flowering, vegetative plants. The corresponding data were obtained by grouping data from the time curves above according to the developmental state instead of the time of harvest. Nodule biomass was more than three times as high in flowering as compared to non-flowering plants. Since senescence was induced on flowering plants, the nodule biomass of the corresponding plants was not significantly different as compared to flowering plants (Table 1). As a marker for nodule senescence, nitrogenase and protein contents were measured. Nitrogenase activity was nearly three times as high in flowering as in vegetative plants. One week after darkening, nitrogenase activity had dropped to very low levels (Table 1). At this stage, nodules had a greenish colour suggesting an advanced proteolyis. The soluble protein content determined after pelleting of bacteroids and cell debris of these nodules had dropped to nearly 25% of the soluble protein content of nodules from flowering, non-senescent plants. Nodules from flowering plants had lower soluble protein contents g-1 FW than nodules from vegetative plants (Fig. 4) confirming former results obtained with soybean (Staehelin et al., 1992). Interestingly, non-soluble protein contents due to bacteroids and cell debris in nodules from all three groups did not differ significantly.
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Survival and re-isolation of bacteria
In order to monitor the integrity of bacteroids and their capacity to redifferentiate, aliquots of nodule extracts were stained with a vital stain for bacteria or plated on a selective medium after convenient dilution.
Over the growth period and following dark-induced senescence the total number of bacteroids counted in nodule extracts was in the order of magnitude of 1010 g-1 FW and did not vary significantly (Fig. 5A). This was also true for the percentage of bacteroids detectable as ‘viable’ upon vital staining immediately after extraction (Fig. 5B). The number of bacteroids with a potential to redifferentiate into growing bacteria (colony forming units, CFUs) was not decreased, but even significantly (P<0.05) increased from c. 3x109 g-1 FW in non-senescing nodules to more than 8x109 in senescing nodules (Fig. 5a). As a consequence, the amount of CFUs, expressed as a percentage of total bacteroids, was significantly (P<0.05) higher in nodules from flowering plants than in those from vegetative plants, and it strongly increased further in the senescent nodules from darkened plants (P<0.01).
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Discussion |
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L. japonicus is effectively nodulated by Rhizobium sp. NGR234 thus confirming previous results (Hussain et al., 1999
). To obtain a significant amount of nodules, however, a growth period of 4 weeks is not enough. Only after 9 weeks does nodulation seem to reach a plateau. Over the whole time period, a high number of colony-forming bacterial units could be isolated from the nodules, indicating the capacity of many bacteroids to redifferentiate into growing and dividing bacteria. Contrary to the view that bacteroids are digested during senescence, the total number of bacteroids extracted from senescent nodules did not decrease, and their ability to redifferentiate appeared to be particularly high, reaching almost 50% of the total count of bacteroids and more than 75% of the bacteroids judged to be viable immediately after extraction. This is in agreement with current evolutionary models suggesting that nitrogen-fixing rhizobia in nodules (bacteroids) should have a higher survival probability, after nodule senescence, than free-living rhizobia in the rhizosphere (Provorov, 1998). Some of the colony-forming bacteria obtained may also represent bacteria from infection threads within the nodules which have not undergone differentiation into bacteroids. However, such infection threads persist to a large extent only in indeterminate nodules, thus insuring the continuous infection of newly differentiated cells (Sanchez et al., 1991; Tsyganov et al., 1998). In determinate nodules, such as the ones formed by L. japonicus, the microsymbiont population is considered to be uniformly differentiated to bacteroids in the mature nodule (Adams and Chelm, 1988). As shown in previous studies (Swaraj et al., 1993), the senescence of L. japonicus nodules could be effectively induced upon darkening. The resulting proteolysis mostly affected soluble, plant-derived proteins thus confirming earlier studies on senescing soybean nodules (Pfeiffer et al., 1983; Sarath et al., 1986). Together with recent results concerning the prevalence of the bacteroid-borne disaccharide trehalose in senescing soybean nodules grown under axenical conditions (Müller et al., 2001), evidence is accumulating that bacteroids maintain to a large extent their viability and their ability to redifferentiate into free-living bacteria during nodule senescence. In fact, these new results obtained with L. japonicus colonized by Rhizobium sp. NGR234 suggest an even higher redifferentiation potential in bacteroids from senescing nodules (Müller et al., 2001). This may be due to a different energy status after a down-regulation of Nif genes encoding nitrogenase, or to a remodelling of cell envelope structures. EM-studies on senescing nodules will clarify this point. Moreover, measurements of potential protease activities and levels of reactive oxygen species in the plant and the bacteroid fractions of senescing nodules will show to what extent the bacteroids are exposed to the lytic conditions of the plant part of the nodule.
Taken together, the system of L. japonicus colonized by Rhizobium sp. NGR234 appears promising for studying the genetic basis of the survival and redifferentiation potential of bacteroids both with respect to the macrosymbiont and to the microsymbiont. Plant mutants affecting the viability of rhizobia will be of great interest for comparative studies with other symbionts (e.g. arbuscular mycorrhiza). Moreover, bacteroid genes with unknown functions expressed in mature nodules (Perret et al., 1999) could be interesting candidates for specific investigations concerning viability and redifferentiation. In a recent study, it has been shown that Rhizobium etli effectively nodulates L. japonicus, but the resulting nodules show early senescence and have a lower bacteroid density on EM-pictures than nodules colonized by M. loti (Banba et al., 2001). Here, studies concerning the redifferentiation of rhizobia after stochastic release from nodules and after nodule senescence will be important to complete the picture and thus will lead to a more general view concerning the evolution of plant–microbe interactions perhaps by generalizing the gene-for-gene-interaction model to the later phases of these interactions.
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Acknowledgments |
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We are indebted to Professor Dr W Broughton (University of Geneva, Switzerland) for providing us with the Lotus japonicus seeds and the Rhizobium sp. NGR234 strain. This work was supported by the Swiss National Foundation.
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Notes |
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1 Present address and to whom correspondence should be sent: Friedrich Miescher Institute, POB 2543, Maulbeerstrasse 66, CH-4002 Basel, Switzerland. Fax: +41 61 697 45 27. E-mail: joachim.mueller{at}unibas.ch
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