Journal of Experimental Botany, Vol. 52, No. 358, pp. 943-947,
May 1, 2001
© 2001 Oxford University Press
Original Papers |
Trehalose becomes the most abundant non-structural carbohydrate during senescence of soybean nodules
Botanisches Institut der Universität, Hebelstrasse 1, CH-4056 Basel, Switzerland
Received 20 November 2000; Accepted 28 November 2000
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Abstract |
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Carbohydrate metabolism and symbiont survival were studied in nodules of soybean (G. max [L.] Merr. cv. Maple Arrow infected with Bradyrhizobium japonicum 61-A-101), induced to senesce simultaneously by application of the photosynthesis inhibitor dichloromethyl urea (DCMU). The plant-borne carbohydrates sucrose and starch started to decline after 2 d and reached background levels after 8 d, in parallel with the decline of nitrogenase. However, the microsymbiont-borne disaccharide trehalose declined only by about 40% and subsequently remained at a constant level of c. 6 mg g-1 dry weight up to 14 d, when nodules softened and decayed. The number of re-isolated viable bacteria was not significantly decreased in senescent nodules as compared to control nodules. These results indicate that during terminal senescence of nodules an appreciable part of the bacteria conserve their trehalose pools and survive.
Key words: Bradyrhizobium, nitrogen fixation, nodulation, photosynthesis, senescence, soybean, symbiosis.
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Introduction |
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The symbiosis between rhizobia and legumes has been extensively studied on a genetic and developmental as well as a biochemical level (see for reviews Sanchez et al., 1991
; Udvardi and Day, 1997). However, most of the research efforts have been focussed on the initial phase of this interaction, namely nodule morphogenesis and the onset of nitrogen fixation. Nodule senescence, occurring for instance upon pod-filling of annual legumes, is less well understood (Vikman and Vessey, 1993a, b, c), although this phase is decisive in the evolution of rhizobium–legume symbiosis (Sprent and Raven, 1992). In current evolutionary models, it is suggested 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).
This is in contradiction to the traditional idea that bacteroids are more or less completely digested during nodule senescence (reviewed in Werner, 1992). These assumptions are based on the following observations. (i) During the late phase of nodulation, proteases are induced in the nodule (Pladys et al., 1991; Vincent and Brewin, 2000). (ii) Chitinase isoforms susceptible to lysozyme activities are induced in mature nodules (Staehelin et al., 1992; Xie et al., 1999). (iii) Nodule senescence is correlated with an increase of reactive oxygen species (Swaraj et al., 1993; Evans et al., 1999), the presence of oxidized proteins and ultrastructural deterioration (Matamoros et al., 1999). (iv) In senescing nodules, the peribacteroid membrane and even the bacteroids tend to lyse (Pladys and Rigaud, 1988; Udvardi and Day, 1997).
However, the survival of bacteria has not been adequately addressed in many studies on nodule senescence (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 (Pfeiffer et al., 1983; Manen et al., 1991). For example, in soybean nodules senescing upon nitrate treatment, the number of viable bacteria recovered from the nodules does not decrease as compared to non-senescing control nodules (Müller et al., 1994b). It is possible that trehalose, a stress protectant synthesized by many micro-organisms and also by rhizobia, is important in their survival.
To get more information about the survival of bacteria and carbohydrate metabolism during nodule senescence, avoiding the rapid inhibition of nitrogenase due to feed-back inhibition as observed upon defoliation (Hartwig et al., 1994), nodulated soybean plants were treated with the photosynthesis inhibitor DCMU, a specific inhibitor of photosystem II taken up rapidly and with good systemic mobility (see for review Arnon, 1977). Nodules were then harvested over a period of 14 d and the levels of the major plant non-structural carbohydrates, sucrose and starch, as well as the major bacterial carbohydrate, trehalose, were monitored. In parallel, the survival of re-isolated bacteria from the nodules was examined.
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Materials and methods |
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Plant material
Soybean (G. max [L.] Merr. cv. Maple Arrow) seeds were surface-sterilized, inoculated with B. japonicum 61-A-101 and cultivated for 4 weeks in Leonard jars as described previously (Müller et al., 1994
a). To initiate the DCMU treatment, the remaining nutrient solution in the lower part of the jar was exchanged for 2.0 l of new solution supplemented with 0.5 ppm DCMU (0.1 ml of a 10 g l-1 stock in methanol) or with 0.1 ml methanol as a control (three replicates per treatment per time point). The plants were harvested at various time points and the nodules were processed for further analyses.
Analytical
Nitrogenase was assayed using entire root systems as described earlier (Müller et al., 1994b).
Prior to nitrogenase measurements, nodule aliquots were harvested from each root system and immediately chilled. Soluble carbohydrates were extracted using methanol (80% w/v), derivatized and analysed by gas chromatography as described previously (Müller et al., 1994a). From the remaining pellets, starch was assayed as described previously (Müller et al., 1994b).
After measuring nitrogenase, nodules were harvested, weighed and chilled for subsequent analysis of proteins. Soluble proteins were determined in the supernatant (according to Bradford, 1976) after homogenization of 1 g nodules in 3 ml Tricine/K+ (50 mM, pH 7.8) supplemented with 1 mM PMSF, 1 mM EDTA and insoluble PVP (1% w/v) and centrifugation at 10000 g for 10 min.
In order to re-isolate bacteria, aliquots of nodules were harvested at the beginning, surface-sterilized by immersion for 10 min in H2O2 (30% v/v) and at the end of the DCMU treatment, homogenized in Eppendorf tubes in 5 vols 0.2 M mannitol buffered with 10 mM phosphate/K+ (pH 7) using a plastic pipette and plated onto 20E-medium amended with 0.2 M mannitol and supplemented with antibiotics as described previously (Müller et al., 1994b). Plates were incubated at 27 °C and colonies were counted after 8 d.
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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Nodule phenotype and nitrogenase activity
Experiments were performed with vigorously growing vegetative soybean plants, 4 weeks after sowing and inoculation with B. japonicum. At this stage, the plants were fully nodulated and effectively fixed atmospheric nitrogen (c. 2.5 nkat g-1 nodule fw; Fig. 1
).
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) or kept as controls (
). Nitrogenase was assayed as acetylene reduction activity (ARA). Mean values ±SE are given for three independent samples for each time point. In some cases, SE were smaller than the symbol size.The herbicide treatment had visible effects on the plants only 9 d after the addition of the herbicide when the first young leaves started to wilt and, after 14 d, all leaves had wilted. Nodules started to soften 12 d after the addition of the herbicide and 14 d was the last time point where nodules could still be collected and processed. Two days later, they were rotten and could not be analysed further.
Nitrogenase activity started to decline only 4 d after the addition of DCMU and had reached background levels 8 d after addition. Control plants, treated with methanol not containing DCMU had nitrogenase values oscillating around 3 nkat g-1 nodule fw (Fig. 1
).
Control nodules had soluble protein contents reaching more than 20 mg g-1 nodule fw. Upon addition of DCMU, these values declined reaching less than 7 mg g-1 nodule fw after 14 d (Fig. 2).
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Non-structural carbohydrates
In order to see whether the drastic decrease of nitrogenase was correlated to a decrease of assimilates potentially available to the bacteroids, nodule sucrose and starch pools were analysed.
At the beginning of the experiment, nodules had starch contents of more than 40 mg g-1 nodule dw. In nodules of control plants, this pool size decreased only slightly during the experiment (Fig. 3A). In DCMU-treated plants, however, the drop of nitrogenase activity and of protein was, as expected, preceded by a drastic decline of starch, already reaching 25% of the control value after 4 d and then declining further to background levels (Fig. 3A).
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Similar observations were made concerning the pool sizes of sucrose. In nodules of control plants, sucrose contents oscillated around 20 mg g-1 nodule dw with a peak of more than 30 mg g-1 nodule after 4 d (Fig. 3B
). In nodules of DCMU-treated plants, sucrose constantly decreased to c. 50% of control after 4 d to reach background values (less than 0.1 mg g-1 dw) after 8 d (Fig. 3B).
To examine whether this decrease of the major plant-borne assimilates was correlated to a similar decrease of bacteria-borne carbohydrate stores, the nodule pool sizes of trehalose (Streeter, 1985; Müller et al., 1995) were analysed. At the beginning of the DCMU treatment the pool sizes of the trehalose did not differ significantly from control values (up to more than 12 mg g-1 dw). Only 10 d after adding DCMU, trehalose values were significantly lower than in controls, but during the experiment, they did not decline to background. Even after 14 d, trehalose values were still about 60% of the control values (Fig. 4).
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Re-isolation of bacteria from nodules
Re-isolation studies were performed with nodules at the beginning of the experiment and after 14 d when nodule senescence was advanced. The number of re-isolated bacteria ranged between 3 and 6x109 CFU g-1 fw. This value was not significantly different in senescing nodules as compared to control nodules (Table 1).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Treatment of nodulated soybean plants by DCMU caused the induction of simultaneous nodule senescence in the whole root system. After 2 weeks, nodules had the typical characteristics of senescence, i.e. a green colour due to haem degradation and a softened consistence. As expected, a decrease of plant-borne assimilates (sucrose and starch), of nitrogenase activity and of soluble proteins were observed during this period.
Interestingly, the number of bacteria that could be re-isolated from nodules was not affected by this artificial senescence. This is in good agreement with previously published results (Müller et al., 1994b) and with unpublished results obtained with Lotus and Vigna nodules (Müller et al., unpublished results). In contrast to plant-borne carbohydrates, the bacterial disaccharide trehalose (Streeter, 1985; Müller et al., 1995) was not depleted in senescing nodules and, with more than 4 mg g-1 dw became the most abundant non-structural carbohydrate at the end of the experiment; other non-structural carbohydrates accounted for less than 1 mg g-1 dw (data not shown). These observations are in good agreement with earlier published results showing that bacteroids are maintained during soybean nodule senescence (as measured by protein contents of bacteroids as compared to the plant cytosol; Pfeiffer et al., 1983). After lysis of the bacteria, trehalose would be accessible to degradation by trehalase, a nodule-stimulated plant enzyme (Müller et al., 1992; Aeschbacher et al., 1999) with high activity levels, also in senescing nodules (data not shown).
The trehalose accumulated in bacteria could protect them against membrane injuries and/or serve as an intermediate energy reserve. It could be synthesized in actively metabolizing bacteroids most likely from glucose-6-phosphate and UDP-glucose issuing from gluconeogenesis fuelled by dicarboxylic acids, the main C-source for bacteroids (Werner, 1992). Recent results indicate, that another pathway involving malto-oligosides produced from glycogen degradation exists in bradyrhizobia (Streeter and Bhagwat, 1999). It is unclear, so far, which pathway is active in bacteroids. Besides nitrogen fixation and gluconeogenesis, organic acids also fuel the biosynthesis of polyhydroxybutyrate (PHB), another main carbon-storage compound, in bacteroids (Udvardi and Day, 1997; Poole and Allaway, 2000). PHB is degraded in senescing nodules as shown by the disappearance of PHB granula in electron micrographs (Pladys and Rigaud, 1988; own observations). Conversely, trehalose seems to be at least partially maintained during senescence. It is expected to be degraded and used as an energy source upon re-initiation of growth by the bacteria, as observed in heat-shocked or starved yeast cells after shifting to permissive growth conditions (Wiemken, 1990). Studies including mutants in trehalose metabolism could provide a better insight how the pools of the major carbon storage compounds are linked and what their respective role in senescing nodules may be.
Taken together, it can be postulated that nodule senescence does not necessarily lead to a decay of the microsymbiont population. A quite substantial amount of the population may still be able to grow and thus be able to transmit selected traits. This would be in accordance with a current evolutionary model (Provorov, 1998). With this perspective, it will be interesting to examine which bacterial genes expressed in the late stage of nodulation (Perret et al., 1999) confer a selective advantage during and after nodule senescence, for example, by performing mixed population studies with the corresponding mutants.
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Acknowledgments |
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This work was supported by the Swiss National Foundation.
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Notes |
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1 To whom correspondence should be addressed. 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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