then translocated to roots via the phloem [12 – 14]. Thus, plant roots may be exposed internally to
elevated levels of amino acids. The pool of amino acids cycling between the root and shoot is consid-
ered to serve as a signal for the plant internal N status [9,15,16]. The amino acids, whether accu-
mulated in the plant internally or supplied exter- nally, usually down regulate the induction of NO
3 −
uptake [2,15,17 – 20] and reduction systems [21 – 26].
Regulation of induction of the NO
3 −
uptake and reduction systems by reduced N compounds has
been attributed to feed-back inhibition [26,27]. Since the induction of both of these systems is
dependent upon the availability of NO
3 −
in the tissue [28], the N metabolites may affect the induc-
tion processes by affecting the availability of the substrate. Sivasankar et al. [26] observed that Gln
and asparagine Asn inhibited the induction of NR activity NRA in corn roots at both low and
high external NO
3 −
supply. They concluded that inhibition was not the result of altered NO
3 −
up- take, but was due to the direct effect of these
metabolites at the transcription level. In contrast, we recently observed that Gln partially inhibited
the induction of NRA in barley roots at lower, but not at higher, exogenous NO
3 −
concentrations M. Aslam, R.L. Travis, and D.W. Rains, unpublished
results. At low NO
3 −
concentrations uptake is mediated by the inducible high affinity transport
system IHATS; whereas at high NO
3 −
most ab- sorption is via the low affinity transport system
LATS [29]. While the induction of IHATS is inhibited by N metabolites [2,15,17,20,30], LATS
is a constitutive system [29,31] and may not be affected by amino acids. The induction of NRA in
both roots [32] and shoots [33] is regulated by NO
3 −
flux. Thus, the inhibition of induction of NRA by N metabolites in barley roots at lower
NO
3 −
supply may be due to decreased NO
3 −
up- take rather than a direct effect on the reduction
system. In the present study the role of glutamic acid
Glu, aspartic acid Asp, Gln and Asn in the induction of the NO
3 −
uptake system and of NRA in barley roots was investigated. These amino
acids were selected because in many plant species they are exported from the leaves to the roots via
the phloem in considerable amounts [34 – 37]. The results show that while these amino acids directly
inhibited enhancement of the activity of NO
3 −
uptake system, independent of NO
3 −
availability, inhibition of the induction of NRA is due to a
decrease in NO
3 −
uptake resulting in decreased NO
3 −
availability.
2. Materials and methods
2
.
1
. Plant culture Barley Hordeum 6ulgare L., var. CM-72 seeds
were germinated and grown hydroponically in 0.2 mM CaSO
4
in the dark as described previously [38]. After 6 days, seedlings were transferred into
N-free, one-quarter strength Hoagland’s solution [39] and placed in a growth chamber Western
Environmental, Napa, CA, USA under continu- ous light at a photosynthetic photon flux density
PPFD of 400 mmol m
− 2
s
− 1
, 25°C and 60 – 65 relative humidity. Incandescent bulbs Sylvania
90A 19TS8MSS, 90 W and cool white fluores- cent tubes Sylvania F96T12 VHOCW, Danvers,
MA, USA supplied light in a ratio of 5:4. The seedlings were either maintained in N-free solution
uninduced seedlings or were induced with NO
3 −
in the absence or presence of amino acids as described below.
2
.
2
. Induction of NO
3 −
uptake and reduction systems
Induction is defined as an increase in enzyme activity above the initial endogenous level. For
induction about 50 seedlings were placed in 51 of one-quarter strength Hoagland’s solution contain-
ing 0.1, 1.0 or 10 mM NO
3 −
and 0 or 1.0 mM amino acids Glu, Asp, Gln, Asn or diazoacetyl-
DL
-norleucine methyl ester DON, an inhibitor of GOGAT, as indicated in the figure legends and
tables. NO
3 −
from the induction solution contain- ing 0.1 mM NO
3 −
was not allowed to deplete below 0.07 mM. In some experiments the seedlings
were pretreated with the amino acids for 6 h before induction with NO
3 −
.
2
.
3
. Measurement of NO
3 −
uptake Net NO
3 −
uptake rates were determined by fol- lowing depletion of NO
3 −
from the uptake solu- tions. All experiments were performed in a
miniature growth chamber set at continuous light
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
