Journal of Experimental Botany, Vol. 49, No. 326, pp. 1545–1554, September 1998
Influence of N and Ni supply on nitrogen metabolism and
urease activity in rice (Oryza sativa L.)
J. Gerendás1,3, Z. Zhu2 and B. Sattelmacher1
1 Institute for Plant Nutrition and Soil Science, University Kiel, D-24118 Kiel, Germany
2 Department of Horticulture, Zhejiang Agricultural University, 310029 Hangzhou, China
Received 8 December 1997; Accepted 14 May 1998
Abstract
Nickel is considered to be an essential micronutrient
in plants because of its role in the metalloenzyme
urease. In order to characterize the metabolic consequences of Ni deprivation, the significance of Ni
supply for growth and N metabolism of rice plants
grown with either NH NO or urea as sole N source
4 3
was evaluated. Growth of plants receiving NH NO was
4 3
not affected by the Ni status, and neither were the
activities of arginase and glutamine synthetase.
However, urease activity was not detectable in leaves
of low-Ni plants, which in conjunction with arginase
action, led to the accumulation of urea in plants grown
with NH NO . Amino acid contents and mineral nutrient
4 3
status (except Ni) were not affected by the Ni
treatment.
Urea-grown Ni-deprived plants showed reduced
growth and accumulated large amounts of urea owing
to the lack of urease activity. These plants were further
characterized by low amino acid contents indicating
impaired usage of the N supplied. They also exhibited
reduced levels of the urea precursor arginine, which
is merely attributed to the overall N economy in these
plant. When urea-grown plants were supplied with
0.5 mmol m−3 Ni in the nutrient solution, the dry
weight and the amino acid N contents were increased
at the expense of the urea contents, indicating efficient use of urea N in Ni-supplemented plants.
A critical Ni concentration in the shoot regarding
dry matter production of NH NO -grown plants could
4 3
not be deduced, while 25 mg Ni kg−1 DW is certainly
inadequate for urea-grown plants. This suggests that
the Ni requirement strongly depends on the N source
employed.
Key words: Amino acids, ornithine cycle, Ni supply, rice,
urea, urease activity.
Introduction
Urease ( EC 3.5.1.5) was one of the first enzymes purified
to homogeneity (Sumner, 1926), but it was not until
1975, that its requirement for Ni was discovered (Dixon
et al., 1975). Early studies on the Ni requirement of
plants focused on the activation of urease, which is still
the only function of Ni proven in higher plants
(Marschner, 1995), and the growth of cell suspensions
and calli of soybean, tobacco and rice on urea-based
media (Polacco, 1977a, b). The influence of Ni supply on
urease activity of intact soybean plants was also demonstrated ( Eskew et al., 1984), and finally Brown et al.
(1987a, b) presented data to suggest that Ni is essential
for barley. In this investigation it was decided to work
with rice (Oryza sativa), another important cereal, in
order to reach a more general conclusion on the Ni
requirement of higher plants.
Although Ni deficiency is unlikely to occur under field
conditions, the increasing use of urea-based fertilizers in
intensive farming and horticulture (Louis, 1992) calls for
more detailed studies on plant urea metabolism, particularly on the grounds that urea-grown plants are highly
sensitive to inadequate Ni supply (Shimada and Matsuo,
1985; Gerendás and Sattelmacher, 1997a). Urea N
acquired by plants is not available for plant N metabolism
unless hydrolysed to CO and NH ( Fig. 1), the latter
2
3
being incorporated by the combined action of glutamine
synthetase and glutamate synthase (Givan et al., 1988;
Marschner, 1995). Glutamate dehydrogenase is not considered to participate in the assimilation of ammonium
3 To whom correspondence should be addressed. Fax: +49 431 880 1625. E-mail: [email protected]
© Oxford University Press 1998
1546
Gerendás et al.
Fig. 1. The production and conversion of urea in plants. GS: Glutamine synthetase, ARGase: Arginase. Modified from Marschner (1995) and
Walker et al. (1985).
in higher plants to any significant extent (Gerendás et al.,
1993; Robinson et al., 1991).
When urease activity is low due to inadequate Ni
supply, urea may accumulate to considerable levels
particularly in urea-treated plants (Gerendás and
Sattelmacher, 1997a, b), which may eventually lead to
alterations in the turnover of ornithine cycle intermediates
( Fig. 1). Therefore the significance of N source and Ni
supply for rice plants was investigated, and special attention was paid to the key enzymes of urea conversion
(arginase, urease), glutamine synthetase and the amino
acid profile.
Materials and methods
Purification of water and nutrients
Water (resistance=10 MOhm cm−1) used for nutrient solutions
as well as nutrient stocks were purified using chelating resin
(Chelex 100, Biorad ), and the purification efficiency was better
than 98% for all K, Ca and Mg stock solutions tested (Gerendás
and Sattelmacher, 1997a).
Plant material and growth conditions
Rice seeds (IR 76) were surface-sterilized in a 0.5% aqueous
solution of Na-hypochlorite for 10 min, rinsed well and
germinated in an incubator at 25 °C on ash-free filter paper,
moistened with 1 mol m−3 CaSO . Four days later seedlings
4
were transferred to a tank containing an NH NO -based
4
3
nutrient solution at half-strength without Ni (Table 1) and
cultured for 11 d. Then four plants each were transferred to 5 l
polyethylene pots, which contained the complete nutrient
solutions as given in Table 1 (treatment initiation). In order to
minimize Ni contamination, the addition rate to the nutrient
solution was adjusted according to weekly analysis. Each
treatment was run with four replicates and the nutrient solutions
were buffered with CaCO to prevent acidification. Experiments
3
were performed in a growth room at a 16 h day length
(300 mmol m−2s−1 photosynthetic active radiation), a temperature regime of 22/17 °C and relative humidity of 70/90% in
order to reduce transpiration.
Table 1. Composition of the nutrient solution (charge balanced
by SO2− and Cl−)
4
Nutrient
mol m−3
Nutrient
mmol m−3
N (NH NO or urea)
4
3
Ka
P
Mg
Cab
6
2
0.25
0.5
2
FeEDTA
Mn
Zn
Cu
Bc
Mod
Nie
45
2
1
0.5
9
0.1
0.5
aPlus additional K from pH adjustments (water, Fe-EDTA).
bPlus additional Ca from CaCO .
3
cGiven as 4.5 times the Mn addition.
dGiven at 1/20 of the Mn addition.
e+Ni treatment only.
Harvest and mineral nutrient analysis
Four weeks after treatment initiation, plants were divided into
shoots and roots. Samples were weighed, transferred into
polypropylene bags, shock frozen with liquid N , and freeze2
dried. After taking the dry weight the material was crushed in
the plastic bag and subsamples ground to a fine powder using
a ball mill (MM2, Retsch). The procedures for nutrient analysis
are described elsewhere (Gerendás and Sattelmacher, 1997a).
Determination of urease, arginase and GS activity
One day before harvest fully expanded leaves as well as apical
root tissue was sampled for enzyme activities. Urease and
arginase ( EC 3.5.3.1) was extracted in a buffer (50 mol m−3
TRIS, 1 mol m−3 EDTA, 5 mol m−3 dithioerythritol, pH 7.5
(HCl )) at a ratio of 6 and 3 ml g−1 FW for leaves and roots,
respectively, using a Potter S homogenizer (Braun, Melsungen,
Germany) cooled with ice. Subsequently the extracts were
centrifuged for 20 min at 15 000 g (4 °C ). Urease activity was
determined by quantifying the release of 14CO from 14C2
labelled urea as described by Gerendás and Sattelmacher
(1997a). Arginase activity was assayed by determining the net
formation of urea from arginine ( Kolloeffel and Stroband,
1973), and urea formed was quantified spectrophotometrically
by the diacetyl carbamido reaction.
Ni supply and N metabolism
Glutamine synthetase (GS, EC 6.3.1.2) was extracted with a
buffer containing 50 mol m−3 TRIS-HCl, 5 mol m−3 EDTA,
and 5 mol m−3 DTE using a Potter S homogenizer cooled with
ice at a ratio of 5 ml g−1 FW. Extracts were centrifuged at
11 000 g for 20 min. GS activity was determined by the
transferase assay (formation of c-glutamyl hydroxamate)
according to Magalhaes and Huber (1991).
Determination of N metabolites
Aqueous extracts (20 mg DW ml−1) were prepared from finely
ground powder, and urea was determined as ammonium after
hydrolysis with urease and nitrate using an automated colorimetric procedure as described by Gerendás and Sattelmacher
(1997a). Amino acids were analysed as o-phthalaldehyde
derivatives using an automated HPLC system (Gerendás and
Sattelmacher, 1997a).
Statistics
When data were subjected to a statistical analysis the treatment
means were tested for significant differences using the LSD test
at P<5% (SAS/STAT, 1988).
Results
Growth
Growth of rice plants was significantly affected by N
source and Ni supply ( Table 2). While Ni had no effect
on the growth of plants with NH NO , dry matter
4
3
production of urea-grown plants was significantly
impaired in the absence of supplementary Ni. All plant
parts participated in this growth response, but since the
shoot DW was particularly responsive the shoot-to-root
DW ratio varied between 1 and 3.9. Within the high-Ni
treatments urea-grown plants produced less dry weight
than those grown with NH NO . Plants grown with urea
4
3
without Ni supplementation were chlorotic, developed
necrotic spots (not shown) and exhibited a much higher
DW percentage than those of the other treatments
( Table 2).
Table 2. Influence of N source and Ni supply on the growth and
the dry matter percentage of rice plants (mean±SD)
Treatment means followed by the same letter are not significantly
different at P<5% according to LSD test.
Plant part
Urea
−Ni
Shoot
Root
Total
Shoot
NH NO
4
3
+Ni
−Ni
Dry weight in g plant−1
1.2±0.1 c
7.6±0.1 b
13.7±0.1 a
1.1±0.1 c
2.5±0.2 b
3.8±0.3 a
2.3±0.1 c
10.0±0.3 b
17.6±0.3 a
Shoot-to-root DW ratio
1.1±0.1 d
3.1±0.2 c
3.6±0.2 b
Dry matter percentage (DW in % of FW )
27.8±0.7 a
17.8±0.2 b
18.9±0.2 b
+Ni
14.2±0.8 a
3.7±0.2 a
17.8±1.0 a
3.9±0.1 a
18.5±0.6 b
1547
Enzyme activities
The urease activity responded drastically to the Ni treatment ( Fig. 2). Activity was particularly high in shoots of
Ni-supplemented plants, about 10 mmol NH h−1 g−1
3
FW, but virtually absent in tissue of low-Ni plants. There
was no Ni–N interaction as the N form supplied had no
significant effect on the urease activity of leaves and roots.
Arginase activity was highly variable and generally low
in roots, but in leaf tissue arginase activity was in the
range of 0.4 to 0.5 mmol urea min−1 g−1 FW except in
low-Ni urea-grown plants (Fig. 3). Generally speaking,
activity of glutamine synthetase behaved similar to the
arginase activity. The urea-grown low-Ni plants expressed
a lower activity in both roots and shoots, while no
differences were observed between the other treatments
( Fig. 4).
N metabolites
Plants grown with urea without supplementary Ni accumulated large amounts of urea in the shoot ( Table 3),
and those grown with NH NO without additional Ni
4
3
also showed a small, but significant accumulation of
endogenous urea in their leaves and roots as compared
to the Ni-supplemented controls. Only traces of nitrate
were found in urea-grown plants, while plants grown with
NH NO accumulated applicable amounts in both shoots
4
3
and roots ( Table 3). The total amino acid N content of
plants grown with NH NO as N source was not influ4
3
enced by the Ni treatment, while Ni supplementation
resulted in high total amino N contents in urea-grown
plants (Table 3). When plants were grown on urea without additional Ni, the amino acid N content was substantially lower in both shoots and roots.
The treatments affected the individual amino acid pattern, which was mostly dominated by asparagine, glutamine and glutamate in both plant parts (Fig. 5). The amino
acid profile of plants grown with NH NO did not
4
3
respond substantially to the Ni supply. Figure 5 shows
that the higher amino N contents of urea-grown high-Ni
plants ( Table 3) results mostly from their higher contents
of asparagine and glutamine. In the shoots the levels of
alanine, GABA, glutamate, and serine are also increased.
The contents of asparagine and glutamine (and glutamate
in shoots) were significantly higher in urea-grown
Ni-supplemented plants than in those grown with
NH NO , while in urea-grown low-Ni plants the levels
4
3
of all amino acids were the lowest found in all of the
treatments. In relation to the Ni-supplemented plants the
contents of the amides asparagine and glutamine
responded most drastically. As far as the urea cycle
intermediates are concerned, only traces of ornithine and
citrulline could be detected in both plant parts. Arginine
was present, but its level in NH NO -grown plants was
4
3
not affected by the Ni treatment, while in urea-grown
1548
Gerendás et al.
Fig. 2. Influence of Ni supply (0 versus 0.5 mmol m−3 Ni added ) on urease activity in leaf blades and roots. All treatment (−Ni versus +Ni)
means (±SD) significantly different at P<5% according to LSD test.
Fig. 3. Influence of Ni supply (0 versus 0.5 mmol m−3 Ni added ) on arginase activity in fully expanded leaf blades and roots (mean±SD).
Fig. 4. Influence of Ni supply (0 versus 0.5 mmol m−3 Ni added ) on GS activity in fully expanded leaf blades and roots (mean±SD).
Ni supply and N metabolism
1549
Table 3. Influence of N source and Ni supply on the urea, the nitrate and the total amino acid N content of rice plants
(mmol g−1 DW, mean±SD)
Treatment means followed by the same letter are not significantly different at P<5% according to LSD test.
Urea
−Ni
Urea content
Shoot
Root
Nitrate content
Shoot
Root
Total amino acid N content
Shoot
Roots
NH NO
4
3
+Ni
−Ni
+Ni
176.6±68.9 a
5.1±0.8 a
3.2±0.7 c
2.7±0.7 ab
5.2±0.7 b
2.3±1.3 b
3.0±0.4 c
1.4±0.8 b
0.5±1.1 b
0.6±0.4 b
0.1±0.2 b
3.8±4.0 b
78.4±6.2 a
102.7±25.9 a
73.8±2.0 a
101.0±10.5 a
183.7±16.7 a
74.0±8.1 a
102.4±18.5 b
37.2±2.8 b
111.0±26.6 b
37.5±6.6 b
26.2±1.7 c
7.4±1.7 c
plants its content showed the same change as was
observed for other amino acids, namely a substantial
reduction in the absence of Ni (Fig. 5).
shows no response to the Ni concentration despite a wide
variation of the latter.
Mineral nutrient concentrations
Discussion
The mineral contents (except Ni) of shoots and roots of
plants grown with NH NO were not substantially altered
4
3
by the Ni treatment ( Table 4). Comparing the nutrient
contents of shoot and root, it is evident that the concentrations of K, P and Zn are similar in the two organs, while
N, Mg, Mn and, particularly, Ca accumulated in the
shoot, while the roots retained Fe and Cu. The Ni
contents were drastically altered by the Ni supply, around
40 versus 1000 mg kg−1 DW in shoots and 80 versus
1660 mg kg−1 DW in roots of low and high-Ni plants,
respectively.
In urea-grown plants the shoot N contents were lower
in low-Ni, but higher in high-Ni plants than in those
receiving NH NO , and a similar trend is seen in the
4
3
root. Several micronutrients (Cu, Zn and Mn) increased
in shoots and roots of low-Ni urea-grown plants, while
the Fe content of the shoot of urea-grown plants was not
affected by the Ni treatment. Again the Ni contents were
significantly reduced by the Ni treatment, and were below
the detection limit (25 mg kg−1 DW ) in shoots of ureagrown plants. In view of the overall variation of the Ni
data, its contents were slightly higher in roots than in
shoots and not affected substantially by the N source.
Significance of Ni for plants grown with NH NO
4 3
Growth of rice plants relying on NH NO was not
4
3
affected by the Ni treatment ( Table 2), which agrees well
with previous results obtained with zucchini (Gerendás
and Sattelmacher, 1997b), but contrasts with the work of
Brown et al. (1987b) who demonstrated, that growth of
barley plants was significantly reduced when the Ni
content of the shoot was reduced below 100 mg kg−1 DW.
The urease activity in leaves and roots of Nisupplemented plants was about 10 mmol NH h−1 g−1
3
FW ( Fig. 2), which agrees well with earlier data for
soybean leaves (Hogan et al., 1983; Krogmeier et al.,
1991), but is slightly higher than previous findings with
zucchini (Gerendás and Sattelmacher, 1997b). Without
supplementary Ni the urease activity was hardly detectable in both tissues sampled ( Fig. 2), which confirms
earlier observations ( Eskew and Welch, 1983; Gerendás
and Sattelmacher, 1997b; Krogmeier et al., 1991) and
agrees with the established Ni requirement of urease
(Dixon et al., 1980).
As the activity of arginase, generating urea ( Fig. 1),
was not affected ( Fig. 3) the very low urease activity in
Ni-deprived rice plants resulted in a small, but significant
urea accumulation ( Table 3), which has been observed
before in zucchini (Gerendás and Sattelmacher, 1997b),
cereals (Brown et al., 1987b) and soybean ( Eskew et al.,
1984). The amount of urea accumulated seems rather
small in relation to the amino acid N contents ( Table 3),
indicating that the flux through the arginase reaction
( Fig. 1) is not substantial (Fig. 3). It is suggested that in
vigorously growing plants, as used in this experiment,
synthesis of arginine may just balance its consumption
for polyamine and protein biosynthesis, while in source
leaves of older plants the larger urea accumulation
Relationship between the Ni status, the dry matter
production and the urease activity
Figure 6 presents the relationship between the Ni status
of the shoots and the shoot dry weight for different N
sources. Performance of plants grown with NH NO was
4
3
not affected by the Ni supply, but urea-grown plants
responded strongly. The relationship between the Ni
status and the urease activity is given in Fig. 7. Here both
urea- and NH NO -grown plants respond in a similar
4
3
fashion. The urease activity in leaves of high-Ni plants
<25 b
1323±341 a
39±22 b
975±147 a
106±10 b
1545±71 a
84±11 b
1660±45 a
207.1±36.1
127.9±34.6
68.4±10.6
75.9±14.1
485.1±59.2
75.2±35.0
31.5±14.7
32.5±20.0
61.3±34.5
64.3±15.0
104.5±34.6
31.7±21.5
956.2±92.5
486.7±42.4
371.3±63.0
439.1±81.1
116.8±16.4
74.6±18.2
51.5±3.8
57.9±8.9
68.7±6.6
56.6±12.6
55.9±4.6
53.0±5.7
24.0±16.8
12.2±8.2
11.5±2.8
6.9±6.4
100.1±34.5
45.1±11.6
32.6±4.9
26.9±4.5
34.3±5.8
32.6±6.6
30.6±3.4
30.5±3.8
2.30±1.03
5.09±2.26
3.52±1.90
3.77±0.94
11.3±2.2
8.5±1.1
8.9±0.3
6.0±4.1
3.2±0.0
3.4±0.1
4.3±0.2
4.4±0.3
4.0±1.1
4.4±0.3
2.4±0.1
2.3±0.3
3.4±0.2
3.8±0.7
3.0±0.3
2.3±0.5
−Ni
+Ni
−Ni
+Ni
−Ni
+Ni
−Ni
+Ni
Urea
Urea
NH NO
4
3
NH NO
4
3
Urea
Urea
NH NO
4
3
NH NO
4
3
Roots
21.6±3.2
36.8±1.0
30.1±1.0
31.6±3.2
6.1±0.3
16.5±1.7
16.8±1.4
17.3±1.2
Shoot
c
a
b
b
b
a
a
a
43.1±5.6
47.7±8.9
43.6±2.1
39.8±3.0
38.7±3.2
53.6±1.9
46.3±6.8
43.3±3.2
Fe
(mg kg−1)
Zn
(mg kg−1)
Cu
(mg kg−1)
Ca
(g kg−1)
Mg
(g kg−1)
P
(g kg−1)
K
(g kg−1)
N
(g kg−1)
Plant
part
Ni
level
N source
Treatment (N source and Ni supply) means of Ni contents followed by the same letter are not significantly different at P<5% according to LSD test.
Mn
(mg kg−1)
Ni
( mg kg−1)
Gerendás et al.
Table 4. Influence of N source and Ni supply on the mineral nutrient concentrations in shoots and roots of rice plants (mean±SD)
1550
indicates where catabolic activity prevails (Gerendás and
Sattelmacher, 1997b). Activity of glutamine synthetase
( Fig. 4) was not influenced by the Ni status of the plant
which is easily explained by the observation that the
dominating ammonium source in leaves of C plants is
3
the photorespiratory N cycle (Givan et al., 1988) and
that the urea flux is low in these as just described.
The contents of the N metabolites were not affected by
the Ni supply in NH NO -grown plants (Figs 3, 4) and
4
3
the amino acid profile was unchanged ( Fig. 5). This
agrees with previous findings in zucchini (Gerendás and
Sattelmacher, 1997b), but contrasts with observations by
Brown et al. (1990) who observed an accumulation of
arginine and nine other amino acids in Ni-deprived barley
plants and argued that this resulted from a disruption of
N metabolism in Ni-deficient barley plants. However, this
is difficult to understand on the grounds of the established
role of Ni in urease function (Fig. 1). At the moment it
is unclear whether these discrepancies result from genuine
differences between the species used, or from insufficient
Ni deprivation, despite the fact that the urease activity
was substantially reduced ( Fig. 2) and the Ni contents
were similar to those reported by Brown et al. (1987b).
The contents of the mineral nutrients in the shoot were
not influenced by the Ni treatment ( Table 4), and most
values indicate medium to high nutrient status as judged
by published values of critical concentrations (Reuter and
Robinson, 1986). In animal nutrition Ni depleted diets
result in impaired absorption of Fe ( Kirchgessner et al.,
1984), but in the results presented here the Fe content of
the low Ni plants actually increased. This response was
not observed in previous work on zucchini (Gerendás
and Sattelmacher, 1997b) and it contrasts with the
reduced Fe content observed in low Ni barley plants by
Brown et al. (1987b). The concentrations of Cu and Fe
were much higher in roots than in shoots and it is
suggested that this represents Fe (and Cu) bound to or
precipitated in or on the root, rather than Fe and Cu
accumulated within the root symplast (Mengel, 1994).
Only a small root to shoot gradient for Ni can be
observed, irrespective of N source and Ni supply
( Table 4), suggesting that Ni is fairly mobile within the
plant. This is supported by earlier work on the Ni mobility
in plants and its accumulation in seeds (Cataldo et al.,
1978; Mishra and Kar, 1974).
Significance of Ni for plants grown with urea
Growth of Ni-deprived urea-grown plants was significantly impaired ( Table 2) and plants were small and chlorotic (not shown), which is in agreement with earlier
observations (Gerendás and Sattelmacher, 1997a, b;
Shimada and Matsuo, 1985). As shown for NH NO 4
3
grown plants Ni deprivation resulted in no detectable
urease activity ( Fig. 2), but urea-grown plants accumu-
Ni supply and N metabolism
1551
Fig. 5. Influence of N source and Ni supply (0 versus 0.5 mmol m−3 Ni added ) on the concentration of selected amino acids in shoots (A) and
roots (B) of rice plants. Bars represent the mean±SD.
Fig. 6. Relationship between the Ni concentration of the shoot and the shoot dry matter production of plants grown with low (#, %) or high
($, &) Ni supply and urea (%, &) or NH NO (#, $) as N source. Ni concentrations below detection limit set to 20 mg kg−1 DW.
4
3
1552
Gerendás et al.
Fig. 7. Relationship between the Ni concentration of the shoot and the urease activity of the leaf blades of plants grown with low (#, %) or high
($, &) Ni supply and urea (%, &) or NH NO (#, $) as N source. Ni concentrations below detection limit set to 20 mg kg−1 DW.
4
3
lated large amounts of presumably mostly exogenous
urea in the shoot, which makes up the difference when
compared to the soluble amino acid N content of
Ni-supplemented urea-grown plants ( Table 3). The magnitude of urea accumulation reached values similar to
those detected in a wide range of plants species grown
under similar conditions (Gerendás and Sattelmacher,
1997a, b) and illustrates the impaired usage of the urea
N provided. Consequently, the N metabolism and thus
the overall growth ( Table 2) was governed by N limitation, which is supported by the low amino acid N pool
( Table 3) and the low amide contents (Fig. 5) of these
plants. One may anticipate that the accumulation of urea
may result in an accumulation of arginine and/or ornithine by feedback inhibition as observed by Shimada
et al. (1980). However, the lower arginine contents of
these plants suggest that their synthesis is governed more
by the availability of N, which supports previous observations (Gerendás and Sattelmacher, 1997a).
The activities of arginase and glutamine synthetase,
generating urea and assimilating ammonium, respectively,
were also reduced (Figs 4, 5), and this is attributed to
the deficiency of metabolically active N in the plants,
rather than to a specific inhibition of arginase activity
due to the high urea concentration ( Table 3). This view
is supported by reduced levels of all enzyme activities
( Figs 3, 4) and N metabolites ( Tables 3, 4) tested, and a
lower shoot-to-root ratio and a higher dry matter percentage ( Table 2) are also typical indications of N deficiency.
As a consequence of the poor growth of urea-grown lowNi plants the mineral nutrient contents were also affected,
and thus the higher contents of Cu, Zn and Mn in the
shoot are merely attributed to a growth related concentration effect.
Ni supplementation improved growth and urease activity of urea-grown plants tremendously ( Table 2), supporting previous reports on the stimulating effects of
moderate Ni supply to urea-grown plants (Gerendás and
Sattelmacher, 1997a; Krogmeier et al., 1991; Shimada
and Matsuo, 1985). The urease activity in leaves of
Ni-supplemented plants was the same for both N sources
( Fig. 2), suggesting that urease activity is expressed constitutively as long as Ni is available. The induction of
functional urease by Ni supplementation markedly
reduced the urea content of the shoot ( Table 3) and made
the urea N accessible for metabolic conversion, which
resulted in the largest amino N pool ( Table 3), the highest
amide ( Fig. 5) and total N contents ( Table 4) in both
shoot and root of all treatments imposed. However,
supplementing the urea-containing nutrient solution with
Ni did not improve the growth to the same level than the
NH NO treatment, which supports earlier observations
4
3
with soybean, tomato (Shimada et al., 1980) and zucchini
(Gerendás and Sattelmacher, 1997b). It is tempting to
speculate that the reduced growth of urea-grown as
compared to NH NO -grown plants is related to the lack
4
3
of osmolytes in the former as suggested for NH+ as
4
compared to NO−-grown plants by Chaillou et al. (1991)
3
and Gerendás et al. (1997). NO− as well as the cations
3
accumulating in maintaining the ionic balance serve as
quantitatively important osmolytes (McIntyre, 1997), but
although the NO− contents were low in urea-grown
3
Ni-supplemented plants ( Table 3), the K contents were
increased in both shoot and root ( Table 4), and the dry
matter percentage of Ni-supplemented plants was similar
to those grown with NH NO ( Table 2). This indicates
4
3
that these plants had no difficulty in regulating their
osmotic relationships and suggests that Cl− may have
Ni supply and N metabolism
compensated for the osmotic potential of NO− in urea3
grown plants (Blom-Zandstra and Lampe, 1983; Zhu
et al., 1997).
Relationship between the Ni status, the dry matter
production and the urease activity
Ni is considered an ultra trace nutrient, and in order to
classify an element essential three criteria have to be met
(Marschner, 1995) namely (1) that a plant cannot complete its life cycle in the absence of the element, (2) that
the given element must not be replaceable and (3) that it
has a specific function. The second and third criteria are
easily met in the case of Ni, but, in the case of ultra trace
nutrients, the first criterion should not be overemphasized
because, due to the highly purified nutrient solutions, it
cannot be excluded that other, as yet unidentified, essential elements are becoming growth-limiting. In this investigation a critical Ni content in plants grown with NH NO
4
3
could not be derived (Fig. 6), and it is suggested that the
critical level of Ni is below 40 mg kg−1 DW, the value
obtained for low-Ni plants ( Table 4). This and previous
results with zucchini (Gerendás and Sattelmacher 1997b)
suggest that the Ni requirement of plants is not uniform
and often much lower than 100 mg kg−1 DW as originally
proposed by Brown et al. (1987b).
Urea-grown plants responded clearly, but unfortunately
intermediate values around the anticipated critical value
of 100 mg kg−1 DW are missing ( Fig. 6). We can only
conclude that Ni contents below 50 mg kg−1 DW are
inadequate for urea-grown plants, but that growth of rice
is rather tolerant to Ni contents in the range of 900 to
1800 mg kg−1 DW. Figure 7 also demonstrates that plant
metabolism, as judged by urease activity, is rather tolerant
to large variations of the Ni content. In view of the
relative changes of the Ni contents and the urease activity
induced by the Ni treatment, it appears that the latter
seems to respond more drastically than the Ni concentration itself, and suggests that the urease activity may serve
as an accurate indicator for the physiologically active Ni
pool in plants.
Conclusions
Plants grown on Ni-deprived urea-based media accumulate urea. This impaired N utilization results in substantial
growth repressions and physiological N deficiency. Rice
plants thus depend on available Ni when growing on
urea-based nutrient media, as has been observed in other
species (Gerendás and Sattelmacher, 1997; Polacco,
1977a). Although the growth of plants with NH NO
4
3
was not affected by the Ni supply, they accumulated
endogenous urea due to arginase action in conjunction
with low urease activity. It is clear that the Ni status of
1553
a plant has significant consequences for the relative
suitability of urea and NH NO as N sources.
4
3
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft. The rice seeds were kindly provided by K
Cassman (International Rice Research Institute, Philippines),
and we wish to thank B Biegler, S Dürre and R EpbinderSchmidt for technical assistance.
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