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Soil Biology & Biochemistry 32 (2000) 1301±1310
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Possible direct uptake of organic nitrogen from soil by chingensai
(Brassica campestris L.) and carrot (Daucus carota L.)
Shingo Matsumoto a,*, Noriharu Ae b, Makoto Yamagata c
a

Shimane Agricultural Experiment Station, Izumo 693-0035, Japan
National Institute of Agro-Environmental Sciences, Tsukuba 305-8604, Japan
c
Hokkaido National Agricultural Experiment Station, Memuro 082-0071, Japan
b

Received 8 April 1999; received in revised form 8 September 1999; accepted 16 February 2000

Abstract
On comparing the nitrogen uptake of four di€erent kinds of vegetables, i.e., pimento, leaf lettuce, chingensai (a kind of
Chinese cabbage), and carrot, from soil to which rapeseed cake (RC) or ammonium sulfate (AS) were applied at the same N
concentration, di€erent N uptake responses were observed. Chingensai and carrot took up more N from the soil with applied
RC than with applied AS. A smaller amount of inorganic N was detected in the soil with applied RC than the one with applied

AS. On the other hand, pimento and leaf lettuce grew better on the soil with applied AS than on that with applied RC. A
possible explanation for the superior N uptake by chingensai and carrot in the soil with applied RC could be the direct uptake
of organic N, especially a protein-like N compound with a uniform MW of 8000±9000 Da, that accumulated in the soil with
applied RC. In order to support this hypothesis, two typical vegetables were examined: chingensai, which responds better to
organic N, and pimento, which responds better to inorganic N. Xylem sap was collected from these plants and analyzed using
size-exclusion high pressure liquid chromatography (HPLC). In xylem sap of chingensai grown in the soil with applied RC, a
peak similar to that found in the soil solutions extracted with phosphate bu€er was detected on the chromatogram, while this
peak was absent from the chromatograms of chingensai grown in inorganic nutrients culture solution. In contrast, there were no
similar peaks for the xylem sap of pimento grown in the soil with applied RC. Further, when chingensai, carrot, and pimento
were cultivated in an N-free medium under aseptic conditions, the N uptake of chingensai and carrot increased with the addition
of a soil solution extracted by phosphate bu€er, while that of pimento did not increase. These results strongly suggest that the
superior N uptake response to the application of organic material in chingensai and carrot might be related to the direct uptake
of organic N from the soil. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Chingensai; Pimento; Size-exclusion HPLC; Protein-like N compound; Xylem sap

1. Introduction
The utilisation of large amounts of nitrogen fertilizer
has become a common agricultural practice for obtaining the high vegetable yields. However, such a practice
has raised many environmental concerns, especially
regarding groundwater pollution due to the leaching of

excess nitrate-N (Singh and Sekhon, 1979; Ritter,

* Corresponding author. Fax: +81-853-24-3342.
E-mail address: smatsu@mx.miracle.ne.jp (S. Matsumoto).

1989). From the viewpoint of the ecient utilization
and management of natural resources, organic vegetable farming using wastes derived from agriculture
and the food industry is becoming popular (Jong and
Kim, 1995). The in¯uence of the addition of organic
materials on N mineralization and dynamics has been
studied over a long period. Most of the studies are
based on the premise that crops take up only inorganic
N released from the organic material. Some authors,
however, have suggested an alternate mechanism. For
example, Mattingly (1973) showed that potato and
sugar beet absorbed N from organic N sources more

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 4 8 - 1

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S. Matsumoto et al. / Soil Biology & Biochemistry 32 (2000) 1301±1310

eciently than barley and wheat. Yamagata et al.
(1996) reported that upland rice took up more N than
maize and soyabean, when a mixture of organic materials (rice bran and straw in a 4:1 ratio) with a high
C-to-N ratio of 20 was applied. Chapin et al. (1993)
reported that sedge grass in tundra soils preferentially
took up amino acid N rather than inorganic N compared with barley. Likewise, Nasholm et al. (1998)
showed that boreal forest plants could take up organic
N by a method in which 13C- and 15N-labeled amino
acids were injected into the organic layer of an old
successional boreal coniferous forest. In our previous
work, we observed that spinach grown on soil supplied
with organic N in the form of rapeseed cake (RC)
took up more N, than when grown on a soil supplied
with inorganic N in the form of ammonium sulfate
(AS), (Matsumoto et al., 1999). All these reports indicate that the N uptake by some crops could not be
explained solely by the amount of inorganic N in the

soil. One of our objectives is, therefore, to compare
the N uptake responses amongst several kinds of vegetable crops.
In a previous study, we applied organic material to
soil and the N status was then examined using sizeexclusion HPLC and SDS-PAGE (Matsumoto et al.,
2000). Results showed that the organic material was
decomposed by soil microorganisms, and the microbial
biomass increased. Some of this microbial biomass was
converted into a decomposition-resistant material with
a uniform molecular weight (8000±9000 Da) and
exhibited some of the characteristics of a protein. This
protein-like N compound was deposited in the soil in
the same manner as when any type of organic material
was applied to any type of soil. This protein-like N
compound existed as a major fraction of the source of
gradually mineralized N and it originated from the
remains of soil microbes (Kai et al., 1973; Marumoto
et al., 1982; Chantigny et al., 1997). Higuchi (1983)
showed that the amount of inorganic N released from
the soil strongly correlated with the amount of a protein-like N compound extracted with 1/15 M phosphate bu€er at pH 7.0. If a plant performs better and
takes up more N in soil amended with organic material, this plant may take up such a protein-like N

compound. Therefore, we attempted to analyze the relationship between the protein-like N compound
extracted from soil with a phosphate bu€er and the
xylem sap of crops grown on a soil with applied RC
or that of crops grown on a solution culture with inorganic nutrient only, using size-exclusion HPLC.
Further, the crops were cultivated on a medium containing the protein-like N compound extracted from
soil as N sources under aseptic conditions in order to
eliminate the microbial rhizosphere e€ects, such as
transportation of these organic N compounds by symbiotic mycorrhizal fungi to the plants or the microbial

mineralization of the protein-like N compound in rhizosphere. In this paper, we discuss the possible direct
uptake of the protein-like N compound by plants.

2. Materials and methods
2.1. Soil culture
Surface soil (0±20 cm) from a ®eld at the National
Institute of Agro-Environmental Sciences (volcanic ash
soil, Tsukuba, Japan) was collected for this experiment. A 4:1 mixture of vermiculite and the Tsukuba
soil (41.7 g C kgÿ1, 3.4 g N kgÿ1 dry soil) adjusted to
pH 6.0 with CaCO3, was used for crop cultivation to
minimize the initial N content. Organic N was applied

at 100 mg N kgÿ1 soil in the form of rapeseed cake
(RC, 50.0 g N kgÿ1, C-to-N ratio: 7.0). Inorganic N
was applied as AS at 100 mg N kgÿ1 soil. The control
did not receive additional N. Single superphosphate
(150 mg P kgÿ1soil) and K2SO4 (100 mg K kgÿ1 soil)
were also supplied to all soils. These soils were incubated at room temperature for 14 days at 60% of their
maximum water-holding capacity. Pimento (Capsicum
annum L. cv. Kyo-midori), leaf lettuce (Lactuca sativa
L. cv. Red ®re), carrot (Daucus carota L. cv. Asubenigosun), and chingensai (Brassica campestris L. cv. choyo No. 2) were germinated on perlite without the addition of any nutrients. Seedlings were transplanted on
11 August 1997 into 500 ml polyethylene pots ®lled
with 400 ml of the incubated soil and grown in a glasshouse. Plants were sampled at 28 days after transplanting (DAT), and the unplanted soil was also collected
for the evaluation of N status at 1, 7, 14, 21, and 28
DAT with three replications. The xylem sap of these
plants was collected at 28 DAT.
2.2. N uptake by crops
Plant samples were dried at 708C for 3 days,
weighed, and ground in a bowl mill for a chemical
analysis. The N concentration was determined using
an automatic NC analyzer (Model NC-80, Sumitomo
Chemicals).

2.3. Inorganic N, amino acids, and protein in unplanted
soils
and NOÿ
Inorganic N (NH+
4
3 ) in the soil was
extracted with 2 M KCl and determined by colorimetric procedures (Auto Analyzer, Technicon Instruments, New York, USA). Amino acids in the soil were
extracted with 0.2 M H2SO4, and the concentration
was determined by the ninhydrin method using L-leucine as a standard (Smith and Stockel, 1954). Protein
in the soil was extracted with 1/15 M phosphate bu€er,

S. Matsumoto et al. / Soil Biology & Biochemistry 32 (2000) 1301±1310

and the concentration was determined by the Lowry
method using egg albumin as a standard (Rej, 1974).
2.4. HPLC analysis of soil solution extracted by 1/15
M phosphate bu€er
Ten grams of the soil sample with 40 ml of 1/15 M
phosphate bu€er (pH 7.0) was shaken for an hour to
extract the protein-like N compound. These extracts

were analyzed by both size-exclusion and ion-exchange
HPLC techniques. The operating conditions for the
size-exclusion HPLC (Shimadzu, LC-6A) were as follows: column, Shimpack Diol 150 (Shimadzu); elution;
50 mM phosphate bu€er containing 0.3 M NaCl at
pH 7.0; ¯ow rate, 2.0 ml minÿ1; sample size, 20 ml;
detector, UV-280 nm. The operating conditions for the
ion-exchange HPLC were as follows: column, IEC
DEAE-825 (Shodex); elution, A: 20 mM Tris±HCl
bu€er at pH 8.2, B: A including 0.5 M NaCl; linear
gradient A to B; ¯ow rate, 1.0 ml minÿ1; sample size:
20 ml; detector, UV-280 nm.

1303

including 50 ml N-free MS liquid medium (Murashige
and Skoog, 1962). Of the MS media added, the control
was N-free MS medium without soil extract, and soil
extract at 10 or 20% in the MS liquid medium on the
basis of volume was contained in the other two media.
The soil extracts were prepared as follows: 200 g of

dry soil was added to glucose (1.6 g) and AS (0.16 g)
in a 500 ml plastic bottle and incubated for 14 days at
60% maximum water-holding capacity. The incubated
soil with 400 ml of 1/15 M phosphate bu€er at pH 7.0
was shaken for 1 h. The soil extract was passed
through the No. 6 ®lter paper, excluding molecular
weights under 3500 Da by dialysis using a cellophane
tube (Spectrapor 3, Wako) in distilled water at 48C.
The dialysis was run, until amino acids and inorganic
N were not detected by the above methods for 3 days.

2.5. Solution culture
Chingensai and pimento seedlings grown for 21 days
under the above conditions described were transplanted into a 50 l plastic container ®lled with Hoagland solution (Aiello and Graves, 1997) and grown for
14 days, the xylem saps of these plants were then collected. This culture solution was continually aerated,
and the pH was adjusted on a daily basis to 6.5.
2.6. Xylem sap
Plant stems were cut at 10 mm above the ground
level. The cutting section was sterilized by wiping with
50% ethanol and then attached to a 1.5 ml plastic

tube with absorbent cotton washed with ethanol.
These procedures were performed at 9:00 h and continued for 8 h, the plastic tube was then removed at
17:00 h. Xylem saps were extracted from absorbent
cotton and analyzed by size-exclusion HPLC under the
same conditions as described above. The elution of the
xylem sap of chingensai from the HPLC analysis was
collected every 20 s, and the N concentration of each
fraction was determined by a micro-nitrogen analyzer
(Mitsubishi Chemical Model TN-05).
2.7. In vitro culture under aseptic conditions
The seeds of pimento, carrot, and chingensai were
surface sterilized for 25 min in 100 ml of sodium
hydrochlorite solution (5%) and washed three times
with sterilized water. Seeds were germinated on sterilized vermiculite. Seedlings were transplanted into 300
ml ¯asks containing 25 g of sterilized vermiculite

Fig. 1. Changes in the concentrations of inorganic-N (A), amino
acids-N (B) and protein-N (C) in the unplanted soil during the
growth period. Vertical bars indicate standard error of mean (n = 3).

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S. Matsumoto et al. / Soil Biology & Biochemistry 32 (2000) 1301±1310

Protein-N concentration of the soil extract was 88.2
mg lÿ1 after dialysis. The puri®ed soil extract was
added to the ¯ask through a membrane ®lter (0.2 mm
Steradisk, Kurabo).
Seedlings were grown for 28 days under a 16 h
photoperiod at 3000 lx supplied by the ¯uorescent
lamps.

and the lowest in the control at 28 DAT. These results
correlated well with the concentration of inorganic N
in the soils undergoing treatment. On the other hand,
despite the lower amount of inorganic N in the soil, N
uptake by carrot and chingensai in the RC treatment
at 28 DAT were 34.3% and 46.3%, respectively,
higher than in the AS treatment (Fig. 2).

3. Results

3.3. HPLC analysis of the soil extract with phosphate
bu€er

3.1. Changes in the concentrations of each N form in
unplanted soil
Inorganic N varied between 28 and 50 mg kgÿ1 in
the control that received no additional N, and between
91 and 133 mg kgÿ1 in the AS treatment. Inorganic N
(41.0±82.5 mg kgÿ1) in the RC treatment increased
gradually with time but failed to equal the amount of
N in the AS treatment (Fig. 1A). The concentration of
amino acids-N in the RC treatment was much higher
than in the AS treatment, although the concentrations
of the amino acids-N were low even in the RC treatment (Fig. 1B). The concentration of protein-N in the
RC treatment was considerably higher than that in the
AS treatment and in the control during the cultivation
period (Fig. 1C).

Fig. 3 shows the chromatograms of the extracts
from the unplanted soils of the AS and RC treatments
at 28 DAT. Both those treatments displayed the same
peak with a retention time of 8.4 min under size-exclusion HPLC and with a retention time of 2.8 min under
ion-exchange HPLC. These results agree with our previous results (Matsumoto et al., 2000). The peak
heights and areas for both HPLC analyses were higher
with the RC than with the AS treatments. This order
corresponds to the concentration of the soil protein
fraction extracted with the phosphate bu€er. The molecular weight of this peak was estimated to be about
8000±9000 Da on the basis of the retention time and
standard molecular weight compounds (Gel-®ltration
compounds, Bio-rad).
3.4. HPLC analysis of xylem sap

3.2. N uptake by the crops in soil culture
N uptake by pimento and leaf lettuce was the highest in the AS treatment followed by the RC treatment

Xylem sap collected from chingensai grown in the
RC treatment showed distinctive major peaks at retention times of 8.4, 8.9, 9.5, 10.6, 11.4 and 12.2 min on

Fig. 2. Nitrogen uptake by pimento, leaf lettuce, carrot and chingensai in soil culture. Di€erent letters within crops represent signi®cant di€erence
(Duncan's new multiple range test, P < 0.05, n = 3). Vertical bars indicate standard error of mean.

S. Matsumoto et al. / Soil Biology & Biochemistry 32 (2000) 1301±1310

the size-exclusion HPLC chromatogram (Fig. 4A). Out
of these peaks, the one at 8.4 min appeared to be identical to the major peak (8.4 min) detected in the soil
extracts. Xylem sap collected from chingensai grown in
Hoagland culture solution containing only inorganic
nutrients did not show the peak at 8.4 min, although it
did show the familiar peaks at 8.9, 9.5, 10.1, 10.6, 11.4
and 12.2 min. No peak at a retention time of 8.4 min
was detected in pimento under the growth conditions
of soil culture or solution culture (Fig. 4B).
Fractions of xylem sap of chingensai grown with the
RC treatment were collected every 20 s from the
elution of size-exclusion HPLC, and the N concentration in each fraction was examined (Fig. 5). The

1305

fraction including the peak at a retention time of 8.4
min was detected as N, so it was concluded that the
8.4 min peak corresponded to a nitrogenous substance
with a molecular weight of 8000±9000 Da.
3.5. N uptake by crops under aseptic conditions
The growth of chingensai and carrot under aseptic
conditions was improved by the addition of the soil
extract. On the other hand, the addition of the soil
extract appeared to have no e€ect on pimento (Fig. 6).
The N uptake of chingensai and carrot increased with
increases in the amount of soil extract added, while
that of pimento did not increase (Table 1).

Fig. 3. Chromatograms of the extracts with 1/15 M phosphate bu€er from the unplanted soils applied with rapeseed cake or AS at 28 days after
transplanting.

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S. Matsumoto et al. / Soil Biology & Biochemistry 32 (2000) 1301±1310

Fig. 4. Size-exclusion HPLC chromatograms of the xylem sap collected from chingensai (A) and pimento (B) grown in the soil applied with rapeseed cake and Hoagland solution.

Fig. 5. Nitrogen concentration in the elution collected every 20 s from the size-exclusion HPLC analysis in xylem sap of chingensai grown in soil
with applied rapeseed cake.

S. Matsumoto et al. / Soil Biology & Biochemistry 32 (2000) 1301±1310

Fig. 6. Photographs illustrating the e€ect of the soil extract with 1/
15 M phosphate bu€er added to the N-free MS medium on the
growth of each of the three vegetable crops under aseptic conditions.
(A) Chingensai, (B) carrot, (C) pimento Right: without soil extract;
Center: soil extract addition of 10% of the MS medium on a volume
basis; Left: soil extract addition of 20% of the MS medium on a
volume basis.

4. Discussion
Under the soil culture conditions, N uptake by
pimento and leaf lettuce was the highest in the AS
treatment, followed by the RC treatment, and lowest
in the control. This tendency corresponded well with

1307

the inorganic N concentration in these soils. In contrast, N uptake by carrot and chingensai in the RC
treatment was higher than in the AS treatment, despite
the lower inorganic N concentration in the RC treatment.
Two di€erent hypotheses are proposed for superior
N uptake by carrot and chingensai with the application of RC. (1). These crops, when compared with
pimento and leaf lettuce, may accelerate N mineralization from organic N in rhizosphere soils through the
activity of protease or other enzymes. However,
Hayano (1983, 1986, 1995) reported signi®cant di€erences in the activity of phosphatase, phosphomonoesterase, phosphodiesterase and b-glucosidase between
soil volumes with or without root systems, hereafter
referred to as `rhizosphere and non-rhizosphere soils',
but the di€erences in protease activity appeared to be
negligible. Kanazawa et al. (1988) also detected no
di€erence in the protease activity between rhizosphere
and non-rhizosphere soils for several crops. (2)
Another possibility is that carrot and chingensai may
take up organic N even more eciently than other
crops. Yamagata et al. (1997a), by examining the
di€erences in the response of cereals to organic N (in
the form of rice bran), demonstrated that upland rice
took up more organic N than maize and soyabean.
However, this was not due to a higher protease activity
in the rhizosphere because the protease activity in
upland rice was lower than that in maize and soyabean. Also, Yamagata et al. (1997b) reported that the
15
N concentration of upland rice was higher than that
of maize and soyabean, when 15N-labeled rice bran
was applied to soils. Inorganic N uptake would reduce
15
N concentration in a crop since inorganic-15N derived from rice bran would be diluted by sucient
inorganic N pool in the soil.
Therefore, they suggested that the higher 15N concentration in upland rice might be caused by uptake of
organic N before mineralization of rice bran. This suggestion was supported by the fact that 15N concentration within crops showed no di€erence when 15Nlabeled ammonium sulfate was applied to soils. Mori
(1986) reported that upland rice and barley absorbed
proteins, such as albumin and hemoglobin in solution
culture. These results imply that plants utilize not only
inorganic N, but also organic N, including molecules
which are bigger than amino acids. Our observation
(Fig. 2) suggested that the preferential uptake of organic N by carrot and chingensai might be related to
their ability to utilize organic N in a soil.
There were larger amino acids-N and protein-N concentrations in the RC treatment than in the AS treatment (Fig. 1B and C). The amount of amino acids-N
in the RC treatment was in the range of 0.37±0.59 mg
N kgÿ1, and that in the AS treatment was 0.27±0.40
mg N kgÿ1 during the growth period (Fig. 1B). These

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S. Matsumoto et al. / Soil Biology & Biochemistry 32 (2000) 1301±1310

Table 1
Nitrogen uptake of vegetable crop species grown on N-free MS medium with or without a soil extract supplement under in vitro conditionsa
Treatment

Control
Soil extract added 10%
Soil extract added 20%
a

Crop species
Pimento (m g N ¯askÿ1)

Carrot (m g N ¯askÿ1)

Chingensai (m g N ¯askÿ1)

73.821.3
69.921.6
76.521.4

23.020.9
69.821.4
167.821.8

54.020.9
86.321.2
153.522.3

Mean2S.E., n = 8.

amounts were extremely low as compared to inorganic-N and protein-N. Nemeth et al. (1988) also
reported that amino acids accounted for only about
3% of soil organic N extracted by electro-ultra®ltration, because those were easily mineralized. If plants
take up amino acids from soil, there must be considerable competition with microorganisms. Yamamuro et
al. (1999) reported that 13C- and 15N-labeled aspartic
acid, glutamic acid, serine and leucine applied to soil
as tracers degraded to inorganic N after 2 or 3 days,
and the proportion of direct uptake of the amino acids
was only about 0.4±1.9% in the tomato plants. Therefore, we considered that the amounts of amino acids
in our study were not enough, if, carrot and chingensai
were only utilizing amino acids preferentially. On the
other hand, we focused on soil organic N, especially
the soil protein fraction, which was the dominant form
of N in the RC-amended soil. It was clear that there
was enough organic N to explain the N absorbed by
chingensai and carrot (34.6±55.9 mg N kg ÿ1 in the
RC treatment, and 18.9±31.0 mg kg ÿ1 in the AS treatment, Fig. 1C).
When the soil extracts at 28 DAT were examined by
size-exclusion and ion-exchange HPLC at 280 nm,
both HPLC chromatograms detected only one major
peak (Fig. 3). Regarding the appearance of a single
peak in the soil extract, we have already reported that
the original peaks of the organic material disappeared
rapidly and formed a major peak at 8.4 min, and
when an antibiotic (chloramphenicol) and organic material were applied simultaneously, the original peaks
remained and the peak at 8.4 min appeared much later
(Matsumoto et al., 2000).
Xylem sap collected from chingensai, which showed
a good response to organic N with the RC treatment
had a peak with the same retention time (8.4 min), as
the soil extract in the size-exclusion HPLC chromatogram (Fig. 4A). Xylem sap from chingensai and soil
extract were mixed, and the solution mixture was analyzed using size-exclusion HPLC. Chromatogram
peaks of both soil extract and xylem sap matched completely (data not shown). The N concentration of the
xylem sap of chingensai grown in the RC treatment
was then analyzed. N was detected in the peak with a

retention time of 8.4 min, and this was assumed to be
a protein-like N compound (Fig. 5). This peak was
absent from the chromatogram of xylem sap from
chingensai grown in a solution with only inorganic
nutrients, while common peaks at 8.9, 9.5, 10.1, 10.6,
11.4 and 12.2 min were present (Fig. 4A). The common peaks might represent amino acids and peptides
that form complexes with polyvalent heavy metal cations during xylem transport (Catald et al., 1988).
Therefore, we believe that this peak with a retention
time of 8.4 min was not produced by chingensai itself.
Carrot, which also showed a good response to organic
N grown in a soil with RC, also produced such a peak
in the chromatogram of xylem sap (data not shown).
In contrast, when pimento was grown in the RC treatment or in culture solution, no such peak was detected
in the xylem sap (Fig. 4B). Pimento showed a poor response to organic N (Fig. 2).
The mineral-containing water in soil is taken into
the root through the surface of the root epidermis.
One of the major routes for transfer of materials
between organs is the vascular bundle, which is composed of xylem and phloem. The xylem consists mainly
of xylem vessels, which form a kind of apoplastic
space, in which the xylem sap ¯ows from the roots to
the shoots. The vital activities of organs depend on the
supply of inorganic and organic compounds from the
xylem sap, which are produced and blended in the
root and transported via xylem vessels (Nooden and
Mauk, 1987). Satoh et al. (1992) showed that several
proteins (i.e., 75,000, 40,000, 32,000, 19,000 and 14,000
Da) were transported into xylem sap in squash seedlings. The proteins might be secreted into the cell wall
or apoplastic space of the cells in the central cylinder
according to their signal sequences and then apoplastically transported to the xylem vessels with the movement of water. Sakuta et al. (1998) reported that
proteins estimated to be 30,000 Da were transported
through the xylem from the root to above ground
organs, such as leaves and shoots in cucumber.
Further, Cleve et al. (1991) reported that poplar storage protein (32,000 Da) could be detected in xylem
sap during the dormant period and especially during
budbreak, and a long distance transport of not only

S. Matsumoto et al. / Soil Biology & Biochemistry 32 (2000) 1301±1310

sugars and amino acids but also protein molecule may
be possible. Though further experiments are needed to
con®rm that the 8.4 min peak (8000±9000 Da) of
xylem sap from chingensai and carrot and that of soil
extract with phosphate bu€er are identical, according
to above reports, our observation suggest that such a
high molecular substance can be taken up by chingensai and carrot, but not by pimento.
Chen et al. (1999) reported that arbuscular mycorrizal fungi (AM) promoted the N uptake of crops.
Amongst those crop species, which showed increased
N uptake on the RC treatment, carrot was considered
to form a bene®cial association with AM fungi, while
chingensai, belonging to Brassica failed to associate
with mycorrhizal fungi (Tompson, 1991). Thus, it is
not clear whether N uptake was stimulated by these
crop species in the RC treatment or by their symbiosis
with AM fungi. So, we cultivated two typical crops
under aseptic conditions in order to eliminate a microbial e€ect, that is, microbial decomposition of protein-like N compounds extracted from a soil and the
direct transport of these substances into these plants
through the hyphae of AM fungi. Under these aseptic
conditions, there was no symbiosis with AM fungi and
the protein-like N compound added to the medium, as
an N source was not mineralized.
The growth and N uptake of chingensai and carrot
(whose xylem saps showed the same peak as the protein-like N compound extracted from soil) were promoted by the addition of soil extract under aseptic
conditions. While those of pimento (whose xylem sap
did not show the same peak as the protein-like N compound in soil) were not promoted (Fig. 6, Table 1).
These results strongly suggest that chingensai and carrot have an ability to take up organic N directly from
the soil extract, while pimento does not have this ability. N uptake response by crop species to organic
amendments seemed to be dependent on this ability.
Two steps must be involved in the absorption of a
protein-like N compound from the soil by chingensai
and carrot. First, chingensai and carrot should have
the ability to solubilize the protein-like N compound
adsorbed by the soil colloid. Hayashi and Harada
(1969) suggested that the soil proteins which resisted
microbial attack were adsorbed by inorganic or organic soil colloids. The action mechanism is not yet
understood; it might, however, be related to root exudates with chelating e€ects such as organic acids (Chaney et al., 1972; Ae et al., 1990). Secondly, protein-like
N compounds from soil must be able to penetrate into
a cell through the cell wall and plasma membrane.
These mechanisms have already been demonstrated in
solution culture with applied haemoglobin. The haemoglobin molecules were taken up through cell membranes by invaginations of the plasmalemma, and they
then moved to vacuoles, where they were digested

1309

(Nishizawa and Mori, 1980). The molecule of the protein like-N compound that we have investigated is
much smaller than haemoglobin, and this phenomenon
may possibly occur in carrot and chingensai. Further
studies of these two mechanisms and how the proteinlike N compound is assimilated by plants are surely
required.

Acknowledgements
We thank Dr. Ancha Srinivasan and Dr. Renfang
Shen for their valuable suggestions and assistance in
preparing this manuscript.

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