Journal of Experimental Botany, Vol. 51, No. 344, pp. 559-566,
March 2000
© 2000 Oxford University Press
Weather and nodule mediated variations in 
13C and 15N values in field-grown soybean (Glycine max L.) with special interest in the analyses of xylem fluids
1 Plant Nutrition Diagnosis Laboratory, National Agriculture Research Center, Kannondai 3-1-1, Tsukuba, Ibaraki 305-8666, Japan
2 Institute of Applied Biochemistry, University of Tsukuba, Tennodai, Tsukuba, Ibaraki 305-8572 Japan
Received 3 March 1999; Accepted 26 October 1999
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Abstract |
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The nodulating soybean (Enrei) and its non-nodulating mutant (EN 1282) were grown in outdoor plots for 2 years (1994: extraordinary dry, high temperature, 1995: ordinary year). Carbon and nitrogen accumulation,
13C and 15N values in plant parts and xylem fluids and 15N values in the water-extractable soil N were analysed throughout the growing period. Plant growth in 1994 was rapid during the early growth stages, but no pods were produced. In 1995, plant growth was normal and pods were formed. The 13C values of the leaves were less negative in 1994 than in 1995 and the nodulated plants showed less negative 13C values than non-nodulated plants in both years. The 13C values of the leaves during the vegetative phase were positively correlated to the leaf N concentrations. Leaf N concentrations in their turn were influenced by nodulation and weather conditions and/or soil available N. The 15N values in the plants and xylem fluids were lower in the nodulated soybean than in non-nodulated soybean in both years, and estimates of the contribution of N2 fixation in nodulated plants based on plant top 15N values were 7–14% in 1994 and 37–63% in 1995. The 13C values of xylem fluids did not differ between nodulated and non-nodulated plants. Thus, the expected contribution by phosphopenolpyruvate carboxylase-mediated CO2 fixation in the root nodules to plant C-incorporation could not have been significant.
Key words:
13C, 15N, nodule CO2 fixation, N2 fixation, soybean.
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Introduction |
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Natural abundances of carbon isotopes (
13C) and nitrogen isotopes (15N) in higher plants can be the signature of their physiology and environmental effects on it (Handley and Raven, 1992
; O'Leary, 1995; Yoneyama, 1995). Soybean (Glycine max L.) is able to fix atmospheric nitrogen by a symbiotic system (nodules) with rhizobial bacteria. N2-fixing soybean plants have different 15N values than non-N2-fixing plants which are totally depending on soil-derived nitrogen (Shearer and Kohl, 1986). The nitrogen acquired by the underground parts is transferred through the xylem to the shoot after partial transformation to ureides or amides. The 15N values in the xylem fluids and plant tissues relate to those of the acquired N with some changes due to N metabolism in the plant (Yoneyama, 1995).
Soybean is a C3-type plant whose 13C values typically are lower than those of C4-type plants (O'Leary, 1995). The 13C value of plant tissues may be affected by environmental factors such as water supply and temperature (O'Leary, 1995) and physiological characteristics such as nodulation (Kumarasinghe et al., 1992) and mycorrhizal infection (Högberg, 1990). The carbon fixed by leaves (source organs) is transported to the sink organs such as fruits and roots including nodules. In N2-fixing soybeans, carbon transferred to the roots and nodules as sucrose returns to the shoot via the xylem after transformation to amino acids and ureides. It was suggested that to produce ureides and amides in the nodules some carbon is fixed by the nodules in supplement to the sugars supplied by the leaves (Vance and Heichel, 1991). This fixation of CO2 by nodules would be catalysed by phosphoenolpyruvate (PEP) carboxylase, which differs from ribulose bisphosphate (RuBP) carboxylase operating in the leaves. The incorporation of carbon fixed by the nodules may also change the 13C value in the nodulated plants, since the 13C values of carbon fixed by PEP carboxylase are expected to be less negative than that of CO2 fixed by RuBP carboxylase (O'Leary, 1995).
It was hypothesized that the presence of nodules not only influences the 15N values of plant parts as a result of fixation of atmospheric nitrogen, but also promotes fixation of CO2 by PEP carboxylase, which should be reflected in lower 13C values of xylem fluids transported from the nodule and possibly also of various plant parts. The order of influence depends on the ratios of soil and atmospheric-derived nitrogen and PEP-carboxylase and RuBP-carboxylase-fixed CO2, any environmental parameters that influence these ratios and the difference in isotopic fractionation between the processes.
This hypothesis was tested by growing nodulating soybeans in an experimental field alongside a non-nodulating soybean line, that functioned as a control without symbiotic nitrogen and carbon fixation in the nodules, and measuring the N and C contents and 13C and 15N values of xylem fluids and plant parts. Since the first year of the trials showed exceptional weather conditions (Table 1), the experiment was repeated under normal weather conditions the following year and the results of the first year were used to study the effects of an extremely dry and sunny summer compared to a normal (control) year.
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Materials and methods |
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Experimental design
The experiment was carried out with a nodulating soybean (cv. Enrei) and a non-nodulating mutant (EN 1282), that served as a control. Because the initial experimental year showed abnormally high temperatures and irradiation levels and low precipitation, the experiment was repeated the following year under more moderate weather conditions (Table 1). The data of the first year now served to check the influences of extreme conditions against a control year, thus creating a bi-factorial experimental design, with nodulation and weather conditions as factors.
Plant cultivation in 1994
On an alluvial soil (N content/1.68 mg g-1 DW, 15N=+6.1
), seeds of the nodulating soybean and the non-nodulating mutant were sown in groups of two seeds per hill with a 30 cm interhill distance and 60 cm between rows of hills on 7 June (designated as day 0). P and K were applied before sowing as described for castor bean (Yoneyama et al., 1998). The xylem fluids and plant samples were harvested on days 44 (xylem fluids only), 52, 58, and 77. One day before each sampling, the soil was irrigated because the 1994 summer had a very low precipitation (Table 1). In the morning (about 10.00 h), the soybean plants were cut at the stem bases (5 cm above the ground surface) and, per sample, 1.5 ml xylem fluid was collected from two hills for triplicate samples and stored frozen. The above-ground parts, separated into leaves and petioles plus stems, were harvested in triplicate, dried in an oven at 70 °C for 1 week and ground to fine powder. Triplicate samples of 200 g fresh soil were taken from the rooting soil zone on the sampling days and stored at 4 °C in darkness.
Plant cultivation in 1995
In the same field used in 1994, the same two soybean lines were sown on 21 June (day 0). The samplings of the xylem fluids, above-ground plant parts separated into leaves, petioles, stems, and pods and rooting soils were conducted in triplicate on days 44, 48, 63, and 78.
Analysis of 13C and 5N values
The C content and 13C value of 1 mg of each ground sample were analysed with an ANCA-SL mass spectrometer (Europa Scientific, UK) in C/N mode. After computing the appropriate sample sizes (containing around 50 µg N), the N content and 15N value in the plant parts were analysed by an ANCA-SL mass spectrometer in single N mode, which includes the removal of CO2 to prevent its interference with the 15N analysis.
For the analysis of 13C and 15N values of xylem carbon and nitrogen, respectively, 0.2–0.3 ml and 0.15–0.30 ml xylem fluids were freeze-dried in tin capsules and analysed by an ANCA-SL mass spectrometer in single C or N mode.
100 g of the stored soil was mixed with 150 ml deionized water, shaken for 60 min and centrifuged at 7500 g for 15 min to obtain the water-extractable N. 100 ml of the supernatant was condensed to 1 ml. 0.05 ml of this concentrate was dried in a tin capsule. The nitrate fraction was obtained by passing the water extractable fraction through a Dowex 50W column. The N content and 15N values in these fractions were determined by an ANCA-SL mass spectrometer in single N mode. 13C and 15N values of samples are expressed by equation [1]:
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15N value of fixed N, the 15N value of fixed N transported from nodules to the host plant, for soybean -1.7, was employed (Yoneyama et al., 1986). |
Results |
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Investigation in 1994
The 1994 summer was extraordinarily hot and dry (Table 1). The accumulation of carbon in the leaves and in the petioles plus stems was rapid before day 52 in both the nodulated and non-nodulated soybeans, but no fruits were produced in either soybean line (Fig. 1
) and the nodule mass of the nodulated soybean was much reduced compared to other years (visual observation). Table 2 shows that the 13C values in the nodulated soybean were less negative than in the non-nodulated plants, and that they increased with the ageing of the plants. The 15N values in the nodulated plants were slightly lower than those in the non-nodulating line and those in the leaves and in the petioles plus stems were similar on days 52 and 58, while on day 77 they had increased in the leaves but decreased in the petioles plus stems. The 15N values for whole plant tops were calculated by isotopic mass balance of the plant parts. The %Ndfa in the nodulated soybean plants was between 7% and 14%.
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) stems plus petioles.
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The concentrations of C and N and the
13C and 15N values of the xylem fluids are shown in Table 3. The xylem fluids from the non-nodulating line were only obtained on day 44 and day 77 because the drought stress reduced the exudation of the xylem fluids more severely in the non-nodulated plants. The C and N concentrations were similar in both soybean types, but the C:N ratios on day 44, when nodules were still abundant on Enrei roots, were lower in the nodulated plants. The 15N values in the xylem fluids were lower in the nodulated plants than in the non-nodulated plants. The calculated %Ndfas for day 44 and day 77 were 35% and 14%, respectively. The concentrations of water extractable N in the soils with nodulated soybean plants were higher on day 58 and day 77, while the 15N values in those soils were lower than in those with the non-nodulated soybean plants (Table 4).
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Investigation in 1995
The 1995 summer showed average weather conditions (Table 1), plant growth was normal and flowers and pods were developed. The early growth of the nodulated soybean line was more vigorous than that of the non-nodulated mutant, while their C content was similar during the late growth period (Fig. 2).
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) petioles, (
) fruits.Table 5
shows the 13C and 15N values in the four plant parts of the nodulated and non-nodulated soybeans. The 13C values in the soybean plants were more negative in 1995 than in 1994 (dry, high temperature) (Table 2). The 13C values in the non-nodulated soybean were more negative than those in the nodulated soybean in all the plant parts, as also observed in 1994 (Table 2). With the ageing of the plants, the 13C values increased in all investigated plant parts of both the nodulated and non-nodulated soybeans. The 13C values in the pods were 1 to 2 less negative than those in the vegetative plant parts. The 15N values in all the plant parts of the nodulated soybean were lower in 1995 than in 1994. In contrast, the 15N values in the leaves of the non-nodulated soybean of 1995 were similar to those in 1994, although the 15N values in the petioles and stems were lower in 1995 than in 1994. The 15N values of the pods were the lowest of all plant parts. The 15N values of the whole plant tops were calculated by isotopic mass balance and, based on them, the %Ndfa in the nodulated soybeans was between 37% and 63% (Table 5).
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The analyses of the C and N concentrations and
13C and 15N values in the xylem fluids are shown in Table 6. The C concentrations in the xylem fluids were similar in the nodulated and non-nodulated soybeans, but the N concentrations were higher in the nodulated soybean line than in the non-nodulated plants. This resulted in lower C:N ratios in the xylem fluids from the nodulated soybean than in those from the non-nodulated plant. The 13C values of the xylem fluids were mostly less negative in 1995 than in 1994. The differences in the xylem 13C values between the nodulated and non-nodulated soybeans were not clear except for day 78 (grain filling). The 15N values of the xylem fluids decreased with the ageing of the plants and were clearly lower in the nodulated soybean than in the non-nodulated plants, suggesting a high abundance of low C : N and low 15N compounds (likely ureides) in the nodulated soybean xylem fluids. The %Ndfa in the nodulated soybean xylem fluids were between 34% and 64%, identical to those calculated based on plant top 15N values (Tables 5, 6).
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The concentrations of water-extractable N and nitrate N in the rhizosphere soils of the nodulated and non-nodulated soybeans are shown in Table 7
. 85–97% of the water-extractable N was nitrate N. The N concentrations between days 48 and 78 were lower in the soil with non-nodulated soybean than in that with the nodulated soybean. The 15N values of the water-extractable N were lower in the soil with the non-nodulated plant than those in the soil with the nodulated soybean on days 63 and 78. The 15N values of soil nitrate N were similar in soils with nodulated and non-nodulated soybeans.
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Discussion |
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The fact that the experiments were carried out in outdoor plots under natural climatic conditions and on a soil that was used previously for agricultural crops implies that the obtained data and differences between the two years are realistic and not extreme results induced by extreme experimental conditions unlikely to occur in the field.
Under contrasting weather conditions, the plants showed different results in growth and C and N physiology as revealed by 13C and 15N values. The less negative 13C values in plants in 1994 compared to those in 1995 (Tables 2, 5) may have been caused by a smaller discrimination of carbon isotopes during CO2 fixation under drought (Farquhar and Richards, 1984). The less negative 13C values in the nodulated soybean compared to the non-nodulated plants in the early stages are in accordance with the previous observation (Yoneyama and Ohtani, 1983). A possible mechanism to explain the less negative 13C value in the nodulated plants is the hypothesis of a contribution of carbon fixed by the PEP carboxylase reaction, which discriminates less for carbon isotopes during CO2 fixation than the RuBP carboxylase reaction (O'Leary, 1995). Nodules were reported to have high activities of PEP carboxylase (Coker and Schubert, 1981). However, the 13C values of xylem fluids in the nodulated soybeans were not higher than those in the non-nodulated plants during the vegetative growth phase with active N2 fixation (Tables 3, 6). This suggests that the contribution of the PEP carboxylase reaction to C-containing compounds transported from the nodules was not significant. Another possible factor influencing the 13C values is the N concentration of the plants. The N concentrations in the leaves of nodulated soybeans were higher than those in the non-nodulated plants in both years. CO2 fixation is more efficient in soybean plants with higher N concentrations (Makino et al., 1988; Sinclair and Horie, 1989) which may reduce internal CO2 concentrations and subsequent isotopic discrimination during fixation. Also as a result of the higher level of solar irradiation in 1994 (Table 1), the intercellular and chloroplast CO2 concentrations (Caemmerer and Evans, 1991) may have become lower in the leaves resulting in a reduction of the discrimination between carbon isotopes. Figure 3 shows the relationship between the N concentration and 13C value of soybean leaves harvested on days 52 and 58 in 1994 and on days 44, 48 and 63 in 1995. (The data for day 77 in 1994 and day 78 in 1995 were not included because plants had lower N concentrations due to senescence.) The results in Fig. 3 clearly indicate that leaves with a high N concentration had less negative 13C values (Pearson Product Moment Correlation, r=0.84, P 0.001). It was reported that the N content (% dry weight) of cladodes of 
-grown Casuarina equisetifolia was the highest among , 
- and N2-grown C. equisetifolia plants while their 13C value was the least negative (Martínez-Carrasco et al., 1998). Such a relationship between N concentrations and 13C values is in accordance with our results. Because of the high correlation between leaf N concentration and 13C, it seems that if water stress caused the less negative 13C values of soybean leaves in 1994 as it did in the experiments of Farquhar and Richards (Farquhar and Richards, 1984), it did so by influencing the leaves' N concentration, possibly through processes which determine N uptake or distribution. However, if the rapid growth and high N accumulation during the early growth period in both nodulated and non-nodulated plants in 1994 is considered, water stress was probably not severe enough to seriously hamper N (mainly soil-N) uptake although N2 fixation was depressed. During the late growth stages, plants were highly stressed in both N2fixation and pod setting, indicating that water stress has become more severe during the growing season. The higher amounts of N per plant in combination with the higher water-extractable N concentrations in the soil show that more N was available in the system in 1994, irrespective of nodulation. At present it seems impossible to conclude whether the higher N-contents in leaves in 1994 were the result of this higher N-availability in the soil, the effects of water stress on N distribution in the plant or both.
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) Nodulated soybean 1994, (•) non-nodulated soybean 1994, (
) nodulated soybean 1995, (The pods are the final sink of N in the plants. The
15N values in the pods were the lowest among the plant parts (Table 5). The N of pods is supplied by both, phloem and xylem fluids. Since the 15N values in the pods were lower than those of the xylem fluids (Table 6), the N transported via the phloem likely had lower 15N values, which was observed before for wheat (Yoneyama et al., 1997) and castor bean (Yoneyama et al., 1998).
The non-nodulated plants absorb soil N, mainly as nitrate (Pate et al., 1993), while the nodulated plants use soil N and N fixed by nitrogenase in the nodules. When the two N sources (soil N and fixed N) have different 15N values, the non-nodulated and nodulated plants will also have different 15N values (Shearer and Kohl, 1986), as observed in this experiment. In soybean, fixed N is transported as ureides from the nodules to the plants through the xylem and these ureides (allantoin and allantoic acid) have low 15N values (-1.4, Bergersen et al., 1988). The N of xylem fluids in the non-nodulated plants is composed only of recently absorbed N and N which has come from the top through the phloem and moves back to the top again (Cooper and Clarkson, 1989). As discussed above, the phloem-derived N probably had lower 15N values than xylem N, in agreement with previous observations (Yoneyama et al., 1997, 1998). Therefore, it is expected that the 15N value of the xylem fluids of both nodulated and non-nodulated plants would be less positive than those of the soil extractable N. Surprisingly, throughout the 1994 investigation and the first two harvests of 1995, the 15N values of xylem fluids (Tables 3, 6) of non-nodulated plants were higher than those of the water-extractable soil N (Tables 4, 7). Higher 15N values of xylem fluids than expected were also observed in the study of wheat plants (Yoneyama et al., 1997). The reason is not clear, but one probability is that the detachment of the top induces an increased proportion in xylem N from N sources (likely ammonia and amino acids) with high 15N values other than soil nitrate. Detailed analysis of xylem N compositions and their 15N values may answer this question.
The main conclusions are that the 13C values of measured xylem fluids show that the hypothesized contribution of CO2fixation by nodule PEP carboxylase could not have played a major role in this experiment. Instead, a close positive correlation between leaf N-concentration and 13C values were found. Weather conditions and/or soil available N and N fixation influenced the N concentrations of plant parts and so the isotopic discrimination of fixed CO2. Specifically, leaf N concentrations were higher in nodulated plants and in the dry year than in non-nodulated plants and in the year with normal precipitation, which resulted in less negative plant 13C values. The 15N values indicated a smaller participation of N fixation in nodulated plants in the year with abnormally high temperatures and irradiation and low precipitation, although the plants total N contents in that year were higher.
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Acknowledgments |
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The authors thank Dr S Akao of the National Institute for Agro-Environmental Sciences, Tsukuba, for supplying of the seeds of a non-nodulating soybean line (EN 1282).
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Notes |
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3 Present address: National Institute of Agro-Environmental Sciences, Kannondai 2–1-1, Tsukuba, Ibaraki 305–8604, Japan.
4 To whom correspondence should be addressed. Fax: +81 298 38 8814. E-mail:yoneyama{at}narc.affrc.go.jp
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