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Quantitative Study for Nitrogen Fixation in Intact
Soybean Plant from PETIS Imaging
S. Ishii a) , N. Suzui a) , S. Ito a) , N. S. Ishioka a) , N. Kawachi a) ,
N. Ohtake b) , T. Ohyama b) and S. Fujimaki a)
a) Radiation-Applied Biology Division, QuBS, JAEA,
b) Graduate School of Science and Technology, Niigata University
The nodule is a symbiotic organ of leguminous plants
with rhizobium. Soybean plants utilize nitrogen (N) fixed
by nodules from atmospheric N2. Until now, 15 N, a stable
isotope, has long been used for studies of N2 fixation.
However, because this method is invasive, it has been
difficult to analyze an instant response to environmental (ex.
temperature, light) changes.
The purpose of our study is to image the N2 fixation
noninvasively and analyze the kinetics quantitatively and by
using nitrogen gas labeled with 13 N (half life: 10 min), a
positron emitting isotope, and PETIS (positron-emitting
tracer imaging system). Previously, we succeeded
development of a new method of production of pure
13
N-labelled nitrogen gas and the imaging of nitrogen
1)
fixation . To know the kinetics of nitrogen fixation rate, it
was required to establish the quantitative method from
PETIS data. In this paper, we report the quantitative
analysis of nitrogen fixation from image data.
We produced 13 N-labeled nitrogen gas, and fed it as a
mixture of N2, O2 and He (10:20:70) to the underground part
of intact nodulated soybean plants for 10 min. Then the
tracer gas was flushed out by flowing fresh ambient air.
The serial images by PETIS were collected for 1 h (360
frames × 10 second).
As a result, serial images of distribution of the fixed 13 N
were obtained (Fig. 1). We quantified nitrogen fixation
rates of assimilated nitrogen in the root nodules using the
PETIS data. Regions were set on the clump of nodules on
the image data and a time-activity curve was generated from
the time-course of radioactivity within the region. The
average value of activity (Bq) in the first 10 frames after the
13
N-labeled nitrogen tracer gas was flushed out was
calculated and then converted into the amount of total
nitrogen (mol N2) using the specific activity of the fed gas
(1.7 × 1011 Bq mol -1 N2). This value indicates the amount
of total nitrogen fixed by the nodules during 10 min of
exposure and was used to estimate the rate of nitrogen
fixation (μmol N2 h -1 ). The rate of nitrogen fixation was
estimated be 0.29 μmol N2 h -1 (Table 1; plant No. 6). The
rates of nitrogen fixation of the six test plants were
-1
estimated to be 0.17 ± 0.10 μmol N2 h in the same way
(Table 1). To analyze the export of nitrogen from the
nodules, a linear fit was carried out on the data points of the
time-activity curve of each sample for 20 min from the end
of flushing out of the tracer gas. The slope of the line
(Bq min -1 ) was converted to the decreasing rate
(μmol N2 h -1 ) using the specific activity. The decreasing
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rates estimated for the six test plants are shown in Table 1.
The large variability in the fixation rates may be attributed
to differences in the size of the plants and⁄or in the number
of nodules.
In conclusion, we estimated nitrogen fixation rates
quantitatively in intact soybean plants.
References
1) S. Ishii et al., JAEA Takasaki Ann. Rep. 2008 (2009)
106.
2) S. Ishii et al., Soil Sci. Plant Nutr. 55 (2009) 660-666.
Fig. 1 Photograph of the tested plant (left) and the
integrated PETIS image of nitrogen fixation in the
same area (right).
Table 1 Estimated values for the rates of nitrogen
fixation and decreasing rate.
Plant No.
Fixation rate
(µmol h -1 )
Decreasing rate
(µmol h -1 )
1 0.08 0.006
2 0.09 0.033
3 0.06