Journal of Experimental Botany, Vol. 53, No. 368, pp. 423-428,
March 1, 2002
© 2002 Oxford University Press
Original Papers |
Short-term metabolic responses of soybean root nodules to nitrate
Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, UK
Received 1 October 2001; Accepted 18 October 2001
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Abstract |
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Soybean (Glycine max L. Merr.) plants exposed to 10 mM KNO3 for a 4 d period were used to test the correlation between nitrogenase activity, gene expression and sucrose metabolism. Nitrate caused the down-regulation of sucrose synthase (SS) transcripts within 1 d, although a decline in nodule SS activity and an increase in nodule sucrose content only occurred after 3–4 d. In a second experiment, plants were exposed to 15N-labelled nitrate for 48 h to determine the time period during which nitrate was taken up, and to relate this to the decline in apparent nitrogenase activity (H2 production in air) and the reduction in SS gene transcript levels. The peak of nitrate uptake appeared to be between 8 h and 14 h whilst apparent nitrogenase activity began to decline at about 17.5 h. The SS mRNA signal declined markedly between 14 h and 24 h. The correlative association of these factors is clear. However, SS activity per se does not appear to be related to the initial decline in apparent nitrogenase activity as a result of nitrate uptake. These findings, therefore, do not support the hypothesis that the regulation of nodule function is mediated by the regulation of SS activity.
Key words: Gene expression, nitrogenase activity, soybean, sucrose metabolism.
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Introduction |
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Metabolism in legume nodules needs to be regulated to ensure that the amount of N2 fixed is co-ordinated with the growth and N requirements of the plant. Nodule N2 fixation, therefore, must be able to respond to nutritional variations and environmental stresses in ways which are interactive with, and responsive to the supply of raw materials (sucrose, water, O2, N2, nitrate, other nutrients). It is now generally established that there are regulatory interactions between photosynthesis, carbohydrate accumulation, root:shoot ratio, and other aspects of growth and nutritional status involving the sensing of sugar, nitrate or other N-compounds (Stitt and Krapp, 1999
). One hypothesis, for which there is substantial indirect evidence, is that N2 fixation in legumes is controlled by a feedback of soluble N compounds from shoots to nodules (see reviews by Parsons et al., 1993; Hartwig, 1998; Serraj et al., 1999). If this is the case, then supply of another N compound such as nitrate (a preferred nitrogen nutrient) is likely to have repercussions in terms of the general metabolic state of the plant. Nitrate reduction in leaves is likely to result in the production of amino acids and amides which are then exported to the rest of the plant, and it seems likely that these would be involved in the suppression of N2 fixation.
In legume nodules it has been proposed that this feedback operates through regulation of the flux of O2 into the nodule by the oxygen diffusion barrier (ODB) (Neo and Layzell, 1997). However, recent papers have questioned whether the ODB is the sole mechanism for the control of nodule function. In some instances, notably in plants exposed to mild drought, experiments indicate that nodules are not just limited by O2; there also appears to be a metabolic limitation (Guerin et al., 1990; Diaz del Castillo et al., 1994). When N2 fixation is reduced as a result of a range of environmental perturbations, raising the external O2 concentration rarely increases fixation back to the values of control plants (Gordon et al., 1997). This implies that, for most stresses, changes in the ODB accounts for only a portion of the reduced nodule activity.
Recently, Gordon et al. showed that N2 fixation is directly correlated with nodule SS activity in soybean plants subjected to a variety of stresses and manipulations (Gordon et al., 1997). The most striking declines in both SS and N2 fixation were in nodules of plants suffering mild drought or treated with 10 mM nitrate or 150 mM salt for 6 d. These treatments also induce severe down-regulation of the SS gene. In a more detailed study of metabolic and gene expression effects during the development of drought it was shown that the decline in SS enzyme activity is correlated with the down-regulation of the nodule SS gene (Gordon et al., 1997). This led to the hypothesis that N2 fixation in soybean nodules is not only mediated by changes in O2 flux, but also by their potential to metabolize sucrose through SS activity. In addition, it was proposed that the response of nodules to environmental perturbations may directly involve the down-regulation of the SS gene.
The present work further tests this hypothesis by analysing short-term changes in metabolic capacity and gene expression in nodules of soybean plants treated with 10 mM nitrate. While nitrate supply cannot be considered to be a stress in the same sense as drought, darkness or shoot removal, it is a nutritional perturbation that has clear impacts on N2 fixation. In the soybean cultivar used in these experiments 10 mM nitrate causes N2 fixation to decline by approximately 45% and 75% after 2 d and 4 d, respectively, while the resistance to oxygen diffusion increases 2–3-fold (Arrese-Igor et al., 1997). Interestingly, however, no effect was found on the amounts of nitrogenase proteins.
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Materials and methods |
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Plant growth and treatments
Soybean plants (Glycine max (L.) Merr. cv. Clarke) were inoculated with either Bradyrhyzobium japonicum (strain RCR 3407, experiment 1) or B. japonicum strain USDA 16 (experiment 2) and grown, one per pot, with a nutrient medium lacking nitrogen (Ryle et al., 1978
) in 0.8 l pots of vermiculite in a Saxcil controlled environment cabinet (25/20 °C day/night temperature, 70% relative humidity, 500 µmol m-1 s-1 PPF, 15 h photoperiod). Experimental treatments were imposed when plants were 5-weeks-old (early flowering stage). In experiment 1, plants were grouped randomly into two treatment sets; control and nitrate. For nitrate-treated plants, 10 mM KNO3 was prepared in the -N nutrient solution and 200 ml applied daily to each pot. Control plants were supplied with the same volume of -N nutrient solution but containing 10 mM KCl. All pots were placed in saucers so that plants had access to the full 200 ml each day. Triplicate control (KCl) plants were harvested on days 0, 2 and 4, while triplicate nitrate-treated plants were harvested on days 1, 2, 3 and 4. Nodules for protein and RNA extraction and for metabolite analysis were picked and collected on ice, surface dried with tissue paper, weighed, frozen in liquid nitrogen and stored at -80 °C.
In experiment 2, plants were exposed to either 10 mM KNO3 or 10 mM KCl for 2 d and the nitrate treatment had 28 atom% enrichment with 15N. Triplicate control and treated plants were harvested after 0, 2, 4, 8, 14, 24 and 48 h. Shoots and roots were analysed for 15N enrichment whilst nodules were treated as above. A further four replicates of both nitrate-treated and control plants were sealed into their pots (Minchin et al., 1983) for continuous gas exchange measurements of their nodulated root systems.
Extraction of host plant proteins
Nodules were homogenized in a mortar and pestle with 50 mM MOPS, 10 mM 2-mercaptoethanol, 4 mM MgCl2, pH 7 at 0–2 °C (5 ml g-1 fresh weight). The homogenate was centrifuged for 30 min at 20000 g, 2 °C. Samples (50 µl) of the supernatant were assayed immediately for phosphoenolpyruvate carboxylase (PEPC) activity, and 1 ml aliquots were desalted by low speed centrifugation (180 g, 1 min) through 5 ml columns of Bio-Gel P6DG (Bio-Rad) equilibrated with 50 mM MOPS pH 7, 4 mM MgCl2 buffer. The desalted extract was used to assay several enzymes: PEPC, sucrose synthase (SS), alkaline invertase (AI), phosphofructokinase (PFK), pyrophosphate fructose-6-phosphate phosphotransferase (PFP), malate dehydrogenase (MDH), pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), glutamine synthetase (GS), glutamine oxoglutarate aminotransferase (GOGAT), and aspartate amino transferase (AAT). All assays were performed at 30 °C as described previously (Gordon et al., 1999). The desalted extract was also used to assay for protein, after trichloroacetic acid precipitation (Lowry et al., 1951) and leghaemoglobin (Appleby and Bergersen, 1980).
Nodule extraction for carbohydrates, amino acids, amides and ureides
Nodules were extracted and assayed for starch, sucrose, glucose, fructose, total amino acids, and ureides as described previously (Gordon et al., 1999).
Extraction and analysis of RNA
RNA was extracted from nodules using a hot phenol method as described earlier (Ougham and Davies, 1990), and separated by 1.5% agarose/formaldehyde gel electrophoresis (Sambrook et al., 1989). The RNA was transferred to nylon membranes (Hybond N, Amersham Life Science, UK) by capillary transfer. The membranes were hybridized with cDNA probes labelled with 32P using the ‘Prime it’ random primer labelling kit (Stratagene, La Jolla, CA, USA). The sucrose synthase probe was a partial cDNA clone isolated from a cDNA library of Lotus japonicus (Skøt et al., 1996). The Lb probe was a cDNA clone of the soybean lba gene (Hyldig-Nielsen et al., 1982). The ENOD2 probe was a cDNA clone from soybean (Franssen et al., 1987). Glutamine synthetase (GS; EC 6.3.1.2) message was detected using a clone isolated from a Phaseolus vulgaris cDNA library (Bennett et al., 1989), while the ascorbate peroxidase (AP; EC 1.11.1.11) cDNA probe was from soybean root nodules (Chatfield and Dalton, 1993). After hybridization the membranes were washed twice for 30 min at 30 °C with 2xSSC, 0.1% (w/v) SDS, and once for 30 min at 65 °C with 0.2xSSC, 0.1% SDS (1xSSC is 0.15 M NaCl, 0.015 M Na3-citrate, pH 7.5).
15N analyses
Shoots and roots were oven-dried at 80 °C for 48 h, weighed and ground to pass through a 0.5 mm mesh in a Glen Creston grinder. Subsamples of approximately 3 mg were then weighed and analysed for total N and 15N enrichment with a Europa Anca 20–20 SL mass spectrometer. Total plant 15N (mg) at each time point was calculated as:
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Root gas exchange measurements
Nitrogenase activity and root respiration of intact plants was continuously determined over a 48 h period as H2 and CO2 evolution, respectively, in air using an open flow-through gas system (Witty and Minchin, 1998) with electrochemical H2 sensors and an infrared gas analyser. The sealed roots were watered (through a funnel in a gas inlet port, with excess liquid drained through a basal hole) with 200 ml of either 10 mM KNO3 (nitrate-treated) or 10 mM KCl (controls) just prior to time 0 and again at 24 h. Thirty minutes before supplying these solutions at 0 h and 24 h the gas phase was switched from air to 79% Ar:21% O2 for a 10 min period to allow for measurements of maximum rates of H2 production. These were used in calculations of electron allocation coefficients (EAC) for N2.
Statistical analysis
All effects discussed in this study are significant at P
0.05 using Duncan's multiple range analysis.
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Results |
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Experiment 1
Enzymes: Among the range of nodule host plant enzymes assayed only SS activity was significantly reduced by the nitrate treatment; it started to decline by day 3 and was significantly lower than the control plant activity by day 4 (Fig. 1
). There was no corresponding increase in AI activity to compensate for the decline in SS activity, and none of the enzymes involved in ammonia assimilation or amino acid metabolism were affected over the time-scale of this experiment (data not shown). There were also no significant increases in the activities of ADH and PDC, which would indicate a change from aerobic to anaerobic metabolism (data not shown).
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, control) for 4 d. Data points are means of three readings; bar=LSD at P=0.05.Nodule metabolites: Nodule sucrose levels remained constant for the first 2 d and then increased on day 3 to become significantly greater than the controls on day 4 (Fig. 2a
). However, starch levels appeared to decline dramatically within 1 d of the nitrate treatment and were significantly lower than the controls on day 2 (Fig. 2b). Nodule ureide levels also decreased during the nitrate treatment (Fig. 2c), indicating an effect on N2 fixation by day 2. However, there was no significant effect on the content of total amino acids (data not shown).
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Protein and leghaemoglobin levels: Protein content was not significantly different in nodules from nitrate-treated (mean value of 22.7±0.6) or controls plants (mean value of 23.5±0.8 mg g-1 fresh weight) at any stage of experiment 1. In addition, Lb levels were unaffected by nitrate treatment in comparison with controls (mean Lb content was 0.227±0.004 mg mg-1 protein).
Transcript levels: The amounts of mRNA encoding GS, AP, ENOD2, and Lb did not change over the 4 d nitrate treatment (data not shown). By contrast, the SS mRNA signal had effectively disappeared within 1 d of supplying 10 mM nitrate, although there was some apparent recovery after 3 d (Fig. 3).
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Experiment 2
A second experiment explored simultaneously the time-courses of nitrate uptake (using 15N labelling), apparent nitrogen fixation (H2 production in air) and SS transcript levels over the first 48 h of nitrate supply. The need for continuous N2 fixation measurements also required a change from the Hup+ve B. japonicum strain RCR3407 to the Hup-ve USDA 16 strain which would allow for monitoring of H2 production as a measure of apparent nitrogenase activity.
During the 48 h exposure of nodulated soybean plants to 15N-enriched 10 mM KNO3 the peak rate of 15N uptake occurred between 8 h and 14 h (Fig. 4). Apparent nitrogenase activities (H2 production) of control and nitrate-treated plants showed similar activities and diurnal rhythms for the first 18 h, with a decline in H2 production during the cooler dark period (Fig. 5). At about 17.5 h, H2 output from nitrate-treated plants became significantly lower than control plants (LSD=0.022 for the period 16–23 h at P=0.05) and by 48 h was approximately 58% of control values. Both control and nitrate-treated plants showed a dip in activity between 23 h and 24 h followed by a recovery period of 3–4 h. This was due to a 10 min exposure to Ar/O2, to measure maximum H2 production, followed by watering with nutrients containing KNO3 or KCl (see Materials and methods). An essentially similar response pattern occurred for total root respiration over the time-course (data not shown). During the 48 h period there was no change in the EAC for N2 for either nitrate-treated or control plants (means of 0.68 and 0.69, respectively). The 42% decrease in apparent nitrogenase activity observed at 48 h with cv. Clarke plus the USDA 16 strain is comparable to the 44% decrease after 2 d nitrate treatment measured for Clarke plus RCR3407 by acetylene reduction (Arrese-Igor et al., 1997).
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Also in agreement with data for the Clarke/RCR3407 symbiosis, the SS mRNA signal in Clarke/USDA 16 nodules declined markedly within 24 h of supplying 10 mM nitrate (Fig. 6
). The additional information provided by this experiment suggests that the SS transcript declined between 14 h and 24 h. This correlates with the timing of the initial decline in nitrogenase activity (Fig. 5
) and with the presence of substantial amounts of absorbed nitrate in the plant (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Sucrose synthase was recently shown to be essential for nitrogen fixation (Gordon et al., 1999
). Therefore, if the activity of this enzyme is reduced as a result of regulatory mechanisms in response to stress or changed nutritional status, this could be a means of controlling nodule function. Previous research on the time-courses of dark-stress and drought treatments in soybean (Gordon et al., 1993, 1997) showed major decreases in SS mRNA levels within 1 d of the beginning of the stress. The subsequent reduction in SS activity paralleled the reduction in N2 fixation activity in the case of drought. However, with dark stress, N2 fixation declined very rapidly and probably reflected the rapid depletion of sucrose (due to lack of photosynthetic activity) rather than changes in enzyme capacity to metabolize it. In the present study, the time-scale of the nitrate-induced depletion of SS transcripts was similar to those found for darkness and drought, whilst the more gradual reduction in SS activity appeared to be delayed compared to the significant decline within 2 d during drought. Furthermore, in the case of nitrate, the decline in nitrogenase activity started within 1 d, compared to 2 d for drought stress (Durand et al., 1987). In the present experiments, there appeared to be no shortage of carbohydrate substrate in nodules; sucrose and hexose levels did not change over the period when nitrogen fixation declined (cf. Vessey et al., 1988; Vessey and Waterer, 1992). Starch levels did decline rapidly, but this mirrors the type of effect catalogued by Stitt and others (Stitt and Krapp, 1999; Geiger et al., 1999) where a shortage of plant nitrogen leads to starch accumulation, while addition of nitrate to N-starved plants results in a rapid remobilization of starch.
Thus, in nitrate-treated plants, changes in nodule SS activity or sucrose levels are not correlated with the initial decrease in nitrogenase activity. Therefore, the universal application of the SS hypothesis (Gordon et al., 1997) has to be questioned, as only in the case of drought is there evidence that the stress-induced decline in SS activity could be the cause of a subsequent decline in nitrogenase activity. However, SS activity, as discussed so far, is ‘maximum catalytic activity’ assayed under optimal in vitro conditions. It is possible that activity in vivo is modified by allosteric effectors and products of the reaction (Morell and Copeland, 1985; Gordon, 1995) and also by phosphorylation/dephosphorylation as proposed before (Shaw et al., 1994; Huber et al., 1996). It has yet to be established whether these factors could have significant effects on in vivo activity under stress or altered nutritional conditions.
An intriguing feature of the present data is that the timing of the down-regulation of the SS gene (between 14 h and 24 h) matches the initiation of the decline in nodule function (approximately 18 h after the initial supply of nitrate) measured by gas exchange. It is possible that nitrate interacts directly to suppress SS gene expression, or that signalling by other N compounds might be involved (Parsons et al., 1993; Hartwig, 1998; Serraj et al., 1999; Stitt and Krapp, 1999). However, the decline in the level of SS mRNA was not part of a universal down-regulation of nodule genes in response to nitrate, since the mRNA levels of Lb, GS, AP, and ENOD2 did not change over the time-scale studied. At this stage it is unclear why the down-regulation of the SS gene parallels the initial reduction of nitrogenase activity when other genes are not affected and SS activity appears to be unrelated to N2 fixation over the first 4 d of nitrate supply. The nitrogen status of plants is associated with changes in growth patterns; shortage of nitrogen results in high root:shoot ratios, while relief of nitrogen limitation results in more rapid shoot growth. Since SS has been strongly implicated with sink strength (Zrenner et al., 1995), it is possible that a general down-regulation of SS may occur in roots as a result of nitrate supply inducing a relative reduction in root growth. Further work will be required to unravel the regulatory interactions between sucrose metabolism and nitrogen fixation in nodules, in addition to the role of SS in the control of root growth.
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Acknowledgments |
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We are grateful to David Layzell for providing the Bradyrhizobium japonicum USDA 16 strain. IGER is funded through BBSRC.
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Notes |
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1 To whom correspondence should be addressed. Fax: +44(0)1970828357. E-mail: frank.minchin{at}bbsrc.ac.uk
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