Journal of Experimental Botany, Vol. 52, No. 355, pp. 277-283,
February 2001
© 2001 Oxford University Press
Original Papers |
Rapid N transport to pods and seeds in N-deficient soybean plants
1 Faculty of Agriculture, University of Niigata, 2-8050 Ikarashi, Niigata-city, Niigata 951-2181, Japan
2 Department of Radioisotopes, Japan Atomic Energy Research Institute, Gunma 370-1207, Japan
3 Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Gunma 370-1207, Japan
4 Central Research Laboratory, Hamamatsu Photonics Co., Shizuoka 434-0041, Japan
Received 31 August 2000; Accepted 21 September 2000
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Abstract |
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Non-nodulated soybean (Glycine max (L.) Merr.) plants were cultivated hydroponically under N-sufficient (5 mM NaNO3) or N-deficient (0.5 mM NaNO3) conditions. 13N- or 15N- labelled nitrate was fed to the cut end of the stems, and the accumulation of nitrate-derived N in the pods, nodes and stems was compared. Real-time images of 13N distribution in stems, petioles and pods were obtained using a Positron Emitting Tracer Imaging System for a period of 40 min. The results indicated that the radioactivity in the pods of N-deficient plants was about 10 times higher than that of N-sufficient plants, although radioactivity in the stems and nodes of N-deficient versus N-sufficient plants was not different. A similar result was obtained by supplying 15
to cut soybean shoots for 1 h. The fact that the N translocation into the pods from fed to the stem base was much faster in N-deficient plants may be due to the strong sink activity of the pods in N-deficient plants. Alternatively, the redistribution of N from the leaves to the pods via the phloem may be accelerated in N-deficient plants. The temporal accumulation of 13 in nodes was suggested in both N-sufficient and N-deficient plants. In one 13 pulse-chase experiment, radioactivity in the stem declined rapidly after transferring the shoot from the 13 solution to non-labelled NO3; in contrast, the radioactivity in the node declined minimally during the same time period. Key words: Soybean, nitrogen deficient, nitrogen requirement, nitrate translocation, PETIS.
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Introduction |
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Normal soybean plants have root nodules which house the microsymbionts Bradyrhizobium or Sinorhizobium. Nodulated plants utilize both combined nitrogen absorbed by the roots and nitrogen fixed by the bacteroids in the root nodules. In soybean plants, the nitrogen fixed from root nodules is transported mainly as ureides (allantoin and allantoic acid) whereas the combined N absorbed by the roots, normally nitrate, is transported as nitrate or assimilated into amino acids, mainly asparagine (Ohyama and Kumazawa, 1979
; Ohtake et al., 1994). Previous 15N tracer experiments (Ohyama, 1983) indicated that a significant portion of N originating from N2 was translocated directly to the pods and seeds, while most of the -N absorbed was first incorporated into the protein fraction of roots and leaves, and only later was gradually transported to the reproductive parts following degradation of vegetative protein for several days after the 15 feeding.
PETIS (Positron Emitting Tracer Imaging System) has been developed as a dynamic image measurement system by detecting a pair of 
-rays emitted from a positron such as 11C, 13N or 18F in real time, and it has been used in plant nutrition research to visualize the absorption, translocation and distribution of tracer elements in plants (Hayashi et al., 1997; Nakanishi et al., 1999; Sato et al., 1999). Sato et al. observed that the N originating from 13N-labelled nitrate supplied to roots was transported into the soybean leaf petiole within 6–10 min based on real-time detection by PETIS (Sato et al., 1999). In other studies using 15N-labelled nitrate, it was found that nitrate absorption in non-nodulated T201 plants was faster than the nodulating T202 line. Also, nitrate absorption and translocation was enhanced by the presence of prior to the 15 feeding.
Because nitrate absorption and metabolism in roots should be affected by the N status of the plants, the fate of nitrate transported in the shoot is difficult to assess unless short incubation times and intact plants are used. Therefore, 13 was applied to the cut basal end of the stem and short-term 13N distribution was determined using PETIS. In addition, a similar experiment was performed using 15N as a tracer for quantitative analysis of label uptake and distribution. The objective of these studies was to examine whether the short-term translocation of nitrate via xylem to the pods is influenced by conditions of N nutrition.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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13
feeding experimentsNon-nodulating T201 soybean plants were cultivated hydroponically with 5 mM
(N-sufficient) or 0.5 mM (N-deficient) in the nutrient solution (Ohtake et al., 1997) and grown in the growth chamber (LX-3000, Taitec Co. Japan) under 12 h light (228 µmol m–2 s–1) at 28 °C, and 12 h dark at 18 °C. At 83 d after planting, the basal part of the stem was cut and 13 solution was immediately supplied to the cut end.
Nitrate labelled with 13N (a positron emitter with a half-life of 10 min) was produced from 16O(p,
)13N reaction, bombarding 6 ml of target water with 1 µA of 20 MeV H+ particles from the AVF cyclotron of Takasaki Ion Accelerators for Advanced Radiation Application. The solution of 13 was purified by passing it through a cation exchange column (Hayashi et al., 1997; Sato et al., 1999). The solution fed to plants was prepared by mixing 5 ml of 13 solution (N-deficient plants: 43 MBq; N-sufficient plants: 61 MBq), 2.5 ml of demineralized water, and 0.8 ml of 10 mM KNO3.
In the second pulse-chase experiment, the solution to be fed was prepared by mixing 5 ml of 13 solution (N-deficient plants: 40 Mbq; N-sufficient plants: 51 MBq), 2.5 ml of demineralized water and 0.8 ml of 10 mM KNO3. After 12 min of 13 feeding, the feeding solution was changed to the non-labelled 1 mM KNO3 solution.
In both experiments, the pods at the second trifoliolate leaf were observed by PETIS. The detection area is a 48x50 mm rectangle, between a pair of Bi4Ge3O12 scintillator arrays coupled to a position sensitive photomultiplier (Hamamatsu Photonics Co. Japan).
15 feeding experiment
The experiment was carried out using 15N labelled (70.7 atm%) sodium nitrate fed to plants for a 1 h period. Ten ml of 5 mM Na15NO3 was supplied to the cut end of the stem. This experiment was conducted in the growth chamber at 28 °C, with 228 µmol m-2 s-1 of fluorescent light (LX-3000, Taitech Co. Japan) with four replications. After 1 h of incubation, each plant was separated into leaves, stems, nodes, pods, and seeds. Each sample of plant parts was frozen in liquid nitrogen and lyophilized. Plant samples were weighed and ground into a fine powder by a vibrating sample mill. Total N content of the ground tissue was determined by the Kjeldahl digestion method.
Fifty mg of each ground sample was extracted with 1 ml of 80% ethanol. The amino acid and nitrate concentrations in the extract were determined by a modified ninhydrin method (Takahashi et al., 1993) and capillary electrophoresis (Sato et al., 1998), respectively.
Another 100 mg of each ground sample was extracted three times with 1 ml of 80% ethanol. The extract was evaporated at 40 °C then dissolved with 0.5 ml of deionized water. The solution was passed through a 0.2 ml volume of cation exchange resin column (Dowex 50, H+ form, 200–400 mesh) and the column was washed twice with 0.25 ml of deionized water. The effluent is referred to as the neutral+anionic (NA) fraction. The cationic fraction (C) was obtained by elution of the column with 1 ml of 2 N HCl.
The 15N abundance was determined by emission spectrometry (NIA-1 15N-analyser, Jasco Co. Japan) as previously described (Ohyama and Kumazawa, 1979).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The images of 13N radioactivity around pod position are shown for both N-deficient and N-sufficient plants after 13
was continuously supplied to the basal cut surface of the stem for 40 min (Figure 1). The radioactivity in the stem was detected within 10 min, after which a strong signal was observed in the node position both in the N-sufficient and the N-deficient plant. While the pod image could be clearly observed in N-deficient plant after 21 min, it was very weak in N-sufficient plant even after 31–40 min.
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Figure 2
shows the quantitative changes in radioactivity in pods, stems or nodes over time. In the pod of the N-deficient plant, the radioactivity increased linearly after 15 min of lag-time and the final value was much higher (about 100 counts) than the radioactivity in the pod of the N-sufficient plant (about 10 counts). In the stem and node of both the N-sufficient and N-deficient plant, the increase in radioactivity was similar from 7 min to 17 min after the start of 13 feeding. After this, radioactivity in stems and nodes levelled off and the quantity of radioactivity in the comparable tissues was similar in the two types of plants.
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The results of the second 13
pulse-chase experiment are shown in Fig. 3. The more active incorporation of 13N into the pod on the N-deficient plant was confirmed in this experiment. Compared with N-sufficient plant, the radioactivity in the pod on the N-deficient plant kept increasing after 10 min until the end of sampling time. The patterns of radioactivity accumulation in stems were somewhat different from those in the first experiment. However, the patterns of radioactivity accumulation in nodes were similar in the N-sufficient and the N-deficient plants.
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A similar experiment was conducted using 15
except that a single feeding period of 1 h duration was employed. The total 15N labelled N in shoots averaged 459 µg N and 287 µg N per plant for N-sufficient and N-deficient plants, respectively. The higher total N acquisition in N-sufficient plants may be due to the higher transpiration rate by the larger leaves on these plants. Table 1 shows the dry weight, nitrogen concentration, the percentage of N from labelled N, %N distribution, and 15N distribution in each part after the application of 15N labelled for 1 h. In N-sufficient plant shoots, the N concentration was higher in seeds, leaves, stems, nodes, and pod walls than in N-deficient plants. The N concentration in leaves, stems and nodes in N-deficient plants was significantly lower than in the N-sufficient plant parts (38%, 23% and 38%, respectively) while the differences in N concentration in pod walls and seeds were relatively smaller in N-sufficient plants (73% and 77% of N-deficient plants for pod walls and seeds, respectively).
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The percentage of N derived from 15
was higher in nodes and leaves than in stems in N-sufficient plants. The pods and seeds in N-sufficient plants accumulated negligible levels of labelled nitrogen supplied to the cut end of stem. In N-deficient plants, percentage of N derived from 15 was higher in all organs than in N-sufficient plants. This was mainly due to the low N concentration in the organs of N-deficient plants. The percentage of total N found in pod walls and seeds was higher in N-deficient plants than in the pod walls and seeds of N-sufficient plants. Also, the proportion of 15N found in pod walls and seeds was over 2% in N-deficient plants within 1 h 15 application, whereas 15N was not detected in pod walls and seeds of N-sufficient plants.
The amino acid and nitrate concentrations in all shoot organs of N-sufficient plants were significantly higher than those of N-deficient plants especially in stems and nodes (Table 2). In N-sufficient plants, nitrate concentration was much higher in stems, nodes and leaves than in pod walls and seeds.
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Table 3
shows the N concentration and percentage of N from 15N labelled in the neutral plus anion (NA) fraction and the cation (C) fraction for each plant part. In the N-sufficient plants, the N concentration in leaves and stems in the NA fraction was almost the same as the C fraction, but the concentration in the C fraction was higher than in the NA fraction in N-sufficient seeds. In N-deficient plants, the N concentration tended to be lower in most fractions except for the seed NA fraction. Concerning the percentage N from 15N labelled nitrate in N-sufficient plants, the NA fraction was higher than the C fraction in leaves and stems, suggesting that nitrate remained in these parts. On the other hand, the N from 15 was not detected in the NA fraction of pod walls and seeds, although it was detected in the C fraction in pods. This suggests that nitrate itself is not readily transported to the pod walls and seed within 1 h under N-sufficient conditions. Concerning the N-deficient plants, the NA fraction of pod walls showed high %N from 15N (3.6%), although it was negligible in seeds (0%). 15N in the cationic fraction in pod walls and seeds in N-deficient plants was detectable following the 1 h labelling period.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The results obtained by 13
feeding experiments indicated that N translocation to the pods from nitrate supplied to the basal stem was very fast in N-deficient plants compared to N-sufficient plants. The major form of nitrogen supplied to soybean seeds is thought to be amides. Glutamine was found to be a major nitrogenous solute released from the soybean seed coat, comprising 52% of the N present, and asparagine was the predominant nitrogenous solute in the embryo (58% of the soluble N) (Rainbird et al., 1984). On the basis of a time-course experiment with pea plants where 15 had been fed to detached shoots, Lewis and Pate suggested that the non-reproductive parts, especially the leaves, are the principal centres of assimilation and that the reduced-N is then transported to the developing seeds (Lewis and Pate, 1973). In soybean plants, Ohyama indicated that a significant portion of N originating from N2 was translocated directly to the pod walls and seeds as well as leaves, while most of the -N absorbed was first incorporated into the proteins of vegetative organs such as roots and leaves; subsequently, N was transported to the reproductive parts as a result of protein degradation for several days following 15 feedings (Ohyama, 1983).
Some part of the absorbed N might be assimilated in leaves and transported to pods in the form of amino acids included in the cation fraction. Asparagine was suggested to be a major compound transported via phloem from soybean leaves (Ohyama and Kawai, 1982). In N-deficient plants, the percentage of N from 15 was significantly higher in the C as well as the NA fraction of stems, pods and seeds than in those fractions in N-sufficient plants. This may be due to rapid export of assimilated N from N-deficient leaves. 15N was detected in the NA fraction of N-deficient pod walls, suggesting that some 15 might be directly transported to the pod walls under N-deficient conditions. The %N from 15 was also higher in the C fraction in pods of N-deficient plants. The N from 15 was detected only in the C fraction and not in the NA fraction of seeds in N-deficient plants suggesting that nitrate was mainly assimilated in leaves and pods and then transported to the seeds.
While the N concentration of shoot organs of N-sufficient plants was higher than those in N-deficient plants, the nitrogen distribution in pod walls and especially in seeds in the N-deficient plants was higher than N-sufficient plants (Table 1
). The effect of N starvation of soybean plants at various stage of growth on seed yield and N concentration of plant parts at maturity was reported some time ago (Streeter, 1978). He indicated that N redistribution within the plant during seed growth should allow for the adjustment of N concentration in seeds even if the supply of N is terminated. Results in this study may support his model and indicate that when the plants suffered nitrogen starvation, plants regulated the nitrogen distribution preferential to seeds, or plants reduced the number of flowers and young pods, to accumulate enough nitrogen in the seeds.
In soybean plants, the temporal storage of nitrogen from 13 was suggested around the node position, based on the observation that 13N activity in nodes did not appear to decrease for a while after changing from 13N solution to non-labelled solution. Also, in barley plants, it has been indicated that [13C] methionine fed to the tip of the absorbing leaf was translocated to the ‘discrimination centre’, which was located at the base of the shoot (Nakanishi et al., 1999). These authors suggested that this discrimination centre plays a vital role in the distribution of mineral elements and metabolites in graminaceous monocots.
To conclude, results in this study show that N-deficient soybean plants rapidly transported the nitrogen from nitrate to the pods, compared with N-sufficient plants. Also, these results suggest that nodes may have some function relating to temporary nitrate accumulation, and may control distribution from stems to petioles versus pods.
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Acknowledgments |
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The authors thank Dr John G Streeter (Department of Horticulture and Crop Science, Ohio State University) for careful reading of the manuscript.
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Notes |
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5 To whom correspondence should be addressed. Fax: +81 25 262 6643. E-mail: ohtake{at}agr.niigata\|[hyphen]\|u.ac.jp
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Abbreviations |
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PETIS, Positron Emitting Tracer Imaging System; NA, neutral plus anionic fraction; C, cation fraction.
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