PPFD 700 mmol m
− 2
s
− 1
, 25°C, and 50 – 60 relative humidity. The growth chamber was part
of a fully automated high-performance liquid chromatography HPLC system fabricated by
Goyal and Huffaker [40]. Uptake was initiated by placing 15 seedlings in a 100 ml glass beaker 50
mm id, 80 mm height containing 60 ml of the uptake solution. The beaker was fitted with a
stainless steel screen about 10 mm above the bot- tom and a magnetic bar was placed below the
screen. The roots were held above the screen. The beaker was placed on a magnetic stirrer for rapid
mixing of the solution. The roots were rinsed for 5 – 10 s in N-free solution containing 0.2 mM
CaSO
4
, before placing them in the uptake solu- tions. The uptake solutions contained 2 mM MES
pH 6.0, 0.5 mM CaSO
4
and 0.1 IHATS range or 1.0 LATS range mM NO
3 −
. The uptake solu- tions were aerated during the measurements. The
first sample was withdrawn by the automated sam- pling system about 2 min after the seedlings were
transferred into the uptake solutions. Thereafter, the system automatically removed 0.5 ml aliquots
at 1.5 min intervals for 12 min for NO
3 −
determi- nation by the HPLC system. Samples taken from
the uptake solutions containing 1.0 mM NO
3 −
required dilution prior to analysis. Those samples were removed manually every 6 min for 30 min.
Cumulative uptake was computed from NO
3 −
de- pletion and solution volume data and uptake rates
were calculated by linear regression analysis of the uptake curves as described by Goyal and Huffaker
[40].
2
.
4
. Measurement of total NRA
2
.
4
.
1
. Preparation of extracts Roots 1.5 – 2 g were homogenized with 4 ml of
extraction buffer g
− 1
in a chilled mortar and pestle with acid-washed sand. The extraction
buffer consisted of 0.05 mM Tris – HCl pH 8.5, 1 mM DTT, 10 mM flavin adenine dinucleotide, 1
m M Na
2
MoO
4
, 1 mM EDTA, and 10 mM leu- peptin [41]. The homogenates were centrifuged at
30 000 g for 15 min, and the supernatants were used for the measurement of NRA, NO
3 −
and NH
4 +
.
2
.
4
.
2
. NRA assay Enzyme activity was assayed in vitro by follow-
ing the reduction of NO
3 −
– NO
2 −
. The assay mix- ture contained 50 mmol potassium phosphate
buffer pH 7.5, 20 mmol KNO
3
, 0.8 mmol NADH, and 0.2 ml of the extract in a final volume of 1.8
ml. The assays were conducted at 28°C for 15 min. Adding 0.1 ml of 1 M zinc acetate terminated the
reaction. Excess NADH was oxidized by phena- zine methosulfate. The NO
2 −
that was formed was determined colorimetrically as described below.
2
.
5
. NO
3 −
, NO
2 −
and NH
4 +
determination NO
3 −
was determined by measuring A
210
after separation by HPLC on a Partisil-10 SAX Phe-
nomenex, Torrance, CA anion exchange column [42]. NO
2 −
was determined by measuring A
540
after color development for 15 min with a 1:1 mixture
of 1 wv sulfanilamide in 1.5 M HCl and 0.02 wv n-naphthylethylenediamine dihydrochloride.
NH
4 +
was determined using a continuous flow system in which the samples reacted with 0.45 M
KOH to form NH
3
. The NH
3
thus generated passed through a Teflon membrane and redis-
solved in H
2
O. The electrical conductivity of the H
2
O was then measured [43]. The experiments were repeated 2 – 3 times and
the results of representative experiments are re- ported. The data are means 9 SE of three repli-
cates. All results are reported on the basis of root fresh weight.
3. Results
3
.
1
. Effect on NO
3 −
uptake system Both Glu and Asp, and their respective amines
Gln and Asn, partially inhibited increase in the IHATS activity when they were supplied with 0.1
mM NO
3 −
in the growth medium Fig. 1. After 6 h of exposure to NO
3 −
, the IHATS was inhibited by 55 – 65. Both amino acids inhibited increase
of IHATS to a greater extent than did their amines Fig. 1. In contrast, when the seedlings
were supplied with 1.0 mM NO
3 −
and uptake rates were determined at 1.0 mM NO
3 −
, Glu and Asp inhibited LATS activity by only 25 Fig. 1. Fig.
2 shows the comparative effect of pretreatment with Glu, Asp, Gln or Asn on the subsequent
enhancement of the IHATS and LATS in the absence of these amino acids. After a 6 h exposure
to NO
3 −
, Glu was the most effective inhibitor of
Fig. 1. Effect of amino acids on the inhibition of IHATS and LATS in roots of intact seedlings supplied with NO
3 −
for 6 h. Seedlings were grown hydroponically in N-free solution for 6
days in continuous darkness followed by 24 h in continuous light. The seedlings were then transferred into the solutions
containing 0.1 or 1.0 mM NO
3 −
and 1.0 mM amino acid. NO
3 −
uptake rates were determined after 6 h as described in Materials and methods. The uptake solutions contained 0.1
IHATS or 1.0 LATS mM NO
3
. Vertical lines above the bars represent + SE.
Fig. 3. Effect of pretreatment with Gln andor DON on the inhibition of IHATS in roots of intact seedlings supplied with
0.1 mM NO
3 −
for 6 h. Experimental procedure was the same as described in Fig. 2, except that the seedlings were pre-
treated with 1.0 mM Gln with or without 0.25 mM DON for 6 h prior to supplying with NO
3 −
. In ‘none’ treatment seedlings were not pretreated. Vertical lines above the bars are
+ SE.
inhibited the increase in the activity of IHATS and further increased the inhibitory effect of the Gln
pretreatment Fig. 3. The inhibitory effect of the Glu or Gln plus
DON pretreatment on the enhancement of IHATS activity may be due to a decreased concentration
of the inducer NO
3 −
in the roots. To clarify this seedlings pretreated with Glu were supplied with
10 mM NO
3 −
. At this high concentration, NO
3 −
uptake is facilitated by LATS [45]. Under these conditions Glu still inhibited the increase in the
NO
3 −
uptake rates Fig. 4A, even though the roots accumulated more NO
3 −
than those exposed to 0.1 mM NO
3 −
in the absence of Glu Fig. 4B.
3
.
2
. Effect on NRA All four amino acids partially inhibited the in-
crease of NRA at 0.1 mM NO
3 −
Fig. 5. The extent of inhibition was similar in each case. Inhi-
bition did not occur when the concentration of NO
3 −
in the external solutions was increased to 10 mM even if the concentration of the amino acids
was increased to 10 mM M. Aslam, R.L. Travis, and D.W. Rains, unpublished results. Pretreat-
ment with amino acids resulted in varying re- sponses. Glu was more effective in inhibiting the
increase of NRA than was Asp; whereas no inhibi- tion occurred in roots pretreated with Gln or Asn,
even at 0.1 mM NO
3 −
Fig. 6. However, when IHATS, followed by Asp and Gln and Asn Fig.
2. The response to Gln and Asn was similar. LATS activity was inhibited only by about 30 by
pretreatment with Glu or Asp Fig. 2. Inhibition of the LATS activity in roots supplied with the
amino acids at 1.0 mM NO
3 −
was similar to that of IHATS in untreated seedlings which were sup-
plied with 0.1 mM NO
3 −
Fig. 1 and Fig. 2. Pretreatment with DON, which prevents the as-
similation of Gln by inhibiting GOGAT [44], also
Fig. 2. Effect of pretreatment with amino acids on the inhibi- tion of IHATS and LATS in roots of intact seedlings supplied
with NO
3 −
. Experimental procedure was the same as in Fig. 1, except that the seedlings were pretreated with the amino acids
1.0 mM for 6 h prior to suppling with 0.1 IHATS or 1.0 LATS mM NO
3 −
for 6 h. Vertical lines above the bars represent + SE.
Fig. 4. Effect of pretreatment with Glu 1.0 mM on the inhibition of IHATS A and NO
3 −
accumulation B in roots of intact seedlings supplied with 0.1 or 10 mM NO
3 −
for 6 h. Experimental procedure was the same as described in Fig. 2.
except that the seedlings were supplied with 0.1 or 10 mM NO
3 −
. One hour prior to the uptake measurements the seedlings, supplied with 10 mM NO
3 −
, were transferred into solutions containing 0.1 mM NO
3 −
to remove excess NO
3 −
form the cell wall and free space as well as to minimize efflux. Vertical lines above the bars are + SE.
Fig. 6. Effect of pretreatment with amino acids on the inhibi- tion of NRA in roots of intact seedlings supplied with 0.1
IHATS range or 10 LATS range mM NO
3 −
for 6 h. Experimental procedure was the same as described in Fig. 2.
NRA was assayed as described in Materials and methods. Vertical lines above the bars are + SE.
NRA was not inhibited Fig. 6. Even the inhibi- tion caused by pretreatment with Gln in the pres-
ence of DON was alleviated at 10 mM NO
3 −
Fig. 7.
3
.
3
. Effect on NO
3 −
and NH
4 +
accumulation The amino acids used in this study had little
effect on NO
3 −
accumulation in roots after the 6 h induction period Table 1. However, about four
times more NO
3 −
accumulated in roots supplied with 10 mM NO
3 −
than in those supplied with 0.1 mM NO
3 −
. On the other hand, NH
4 +
accumulation was affected by amino acid treatment. About two
DON was included in the pretreatment media, Gln significantly inhibited the increase in NRA Fig.
7. Pretreatment with DON alone also inhibited the increase in NRA Fig. 7. When pretreated
roots were supplied with 10 mM NO
3 −
, increase in
Fig. 5. Effect of amino acids on the inhibition of NRA in roots of intact seedlings supplied with 0.1 IHATS range or
10 LATS range mM NO
3 −
. Seedlings were grown hydropon- ically in N-free solution for 6 days in continuous darkness
followed by 24 h in continuous light. The seedlings were then transferred to the nutrient solutions containing 0.1 or 10 mM
NO
3 −
and 0 or 1.0 mM amino acids in one-quarter strength Hoagland’s solution and placed in continuous light. After 6 h
NRA was assayed as described in Materials and methods. Vertical lines above the bars are + SE.
Fig. 7. Effect of pretreatment with Gln andor DON on the inhibition of NRA in roots of intact seedlings supplied with
0.1 IHATS range or 10 LATS range mM NO
3 −
for 6 h. Experimental procedure was the same as described in Fig. 3.
NRA was assayed as described in Materials and methods. Vertical lines above the bars are + SE.
Table 1 Effect of amino acids 1 mM on the concentrations of NO
3 −
and NH
4 +
in roots of intact seedlings supplied with 0.1 or 10 mM NO
3 −
for 6 h
a
Root [NO
3 −
] Amino
Root [NH
4 +
] mmol g
− 1
mmol g
− 1
acid 10 mM
0.1 mM 0.1 mM
10 mM 10.23
None 39.10
1.15 9 0.02 0.95
9 0.05
9 0.87
9 0.26
b
4.82 9 0.05 37.84
4.84 8.89
Glu 9
0.22 9
1.22 9
0.05 Asp
37.84 8.83
4.41 5.70 9 0.03
9 0.14
9 0.27
9 1.22
43.13 8.82 9 0.16
11.34 7.85
Gln 9
0.33 9
1.73 9
0.09 Asn
40.48 8.36
10.95 9.23 9 0.21
9 0.23
9 .0.87
9 0.17
a
For experimental procedure see Fig. 1.
b
Each value represents the mean 9 SE. Table 3
Concentrations of NO
3 −
and NH
4 +
in roots of intact seedlings pretreated with Gln in the presence or absence of 0.25 mM
DON for 16 h and then supplied with 0.1 or 10 mM NO
3 −
for 6 h
Pretreat- Root [NH
4 +
] Root [NO
3 −
] mmol g
− 1
ment mmol g
− 1
0.1 mM 10 mM
0.1 mM 10 mM
1.21 1.18
9.79 30.73
None 9
0.13 9
0.30
a
9 0.16
9 0.74
1.75 2.12
28.95 6.25
Gln 9
1.15 9
0.12 9
0.09 9
0.11 3.58
DON 25.32
1.09 1.25
9 0.35
9 0.45
9 0.11
9 0.06
1.52 26.25
Gln 1.82
2.98 +
DON 9
0.35 9
0.10 9
0.13 9
0.15
a
Each value represents the mean 9 SE.
or absence of Gln, decreased NO
3 −
accumulation at 0.1 mM NO
3 −
but had little effect on NH
4 +
accumulation Table 3.
4. Discussion
