Journal of Experimental Botany, Vol. 47, No. 297, pp. 485-495, April 1996
Journal of
Experimental
Botany
Xylem exudation is related to nitrate assimilation
pathway in detopped maize seedlings: use of nitrate
reductase and glutamine synthetase inhibitors as tools
Laure Barthes 1 , Eliane Deleens, Agnes Bousser, Jackson Hoarau and Jean-Louis Prioul
Laboratoire Structure et Metabolisme des Plantes, CNRS URA 1128, Institut de Biotechnologie des Plantes,
Bat 630, Universite de Paris-Sud, Centre d'Orsay, F-91405 Orsay Cedex, France
Received 29 September 1995; Accepted 12 January 1996
Abstract
Introduction
The xylem exudation of detopped 7-d-old seedlings of
Zea mays L. doubled when KCI was present in the root
medium compared to seedlings maintained on water.
It was further enhanced when KCI was replaced by
nitrogen compounds such as nitrate, ammonium and
glutamine. The role of the nitrate assimilation pathway
on the enhancement of xylem exudation rate was
investigated using tungstate, an inhibitor of nitrate
reductase (NR) activity, and phosphinothricin or
methionine sulphoximine, inhibitors of glutamine
synthetase (GS) activity. The sap levels of NO3 , NH4+,
glutamine, and asparagine was used to ascertain the
in vivo inhibition of both enzymes. The tungstate
effects were also checked by measuring leaf in vitro
NR activity and NR protein content. Xylem exudation
rate of detopped seedlings fed with KN0 3 decreased
when the nitrate assimilation pathway was blocked
either at the NR or at GS sites. This decrease was
prevented when urea (acting as NH4+ supply) was given
simultaneously with tungstate. KN0 3 does not act directly on exudation, but through the involvement of
NH4+. The involvement of glutamine was also shown
since GS inhibition resulted in a cancellation of the
enhancing effect of KN0 3 on exudation. As change of
exudation rate was not linked to change in sap osmolarity, it is assumed that the assimilation chain could
modify root water conductance. The role of glutamine
was discussed.
The translocation of nitrate from root to shoot is related
to water transport. This transport still occurs when water
movement is driven by the process named root pressure
which causes xylem exudation in detopped plants.
Minshall (1964, 1968) reported an enhancement of the
xylem exudation rate by nitrate or urea supply in root
medium on detopped tomato plants. The nitrate positive
effect on exudation could either be due to an increase of
osmotic potential difference between root medium and
xylem vessels or to an increase of root hydraulic conductance. This work reassessed these relationships on young
seedlings of maize (Barthes et al., 1995a) and wheat
(Barthes et al., 19956): as the total osmoticum in the sap
can not explain the variations in exudation rate, it is
assumed that nitrate induced the enhancement of exudation rate via an increase of hydraulic conductance.
Whatever the actual causes of water flux enhancement
by KNO3, it seems that the entire assimilation pathway
would be linked to an increase of xylem flux. This
assumption is supported by two facts, (i) urea, considered
as an ammonium supplier, had a similar effect to KNO3
(Minshall 1964, 1968; Barthes et al., 1995a) and (ii) the
KNO3 effect on exudation was prolonged even after it
was removed from the root medium (Barthes et al.,
1995a). The hystereris effect of KNO3 could originate
from the endogenously stored anions in the root tissue.
But, does it act directly or indirectly via reduced forms
originating from its assimilation pathway? The similar
effect of urea speaks in favour of an indirect effect of
nitrate. An attempt to establish such a correlation between
exudation and nitrate reduction was reported by Aschroft
Key words: Exudation, maize, nitrate, conductance, NR,
GS.
1
To whom correspondence should be addressed. Fax: +33 1 69 33 64 24.
Abbreviations: PPT, phosphinothricin; MSO, methionine sulphoximine; NR, nitrate reductase; GS, glutamine synthetase.
© Oxford University Press 1996
486
Barthes et al.
et al. (1972) who used detopped tobacco plants. Although
they failed to observe participation of nitrate reductase
(NR, EC 1.6.6.1) in the process, they clearly showed that
nitrate-dependent exudation might be increased by the
addition of glucose. Touraine et al. (1990) explained the
positive correlation between nitrate uptake in root and
nitrate reduction in shoot via the role of the carboxylate
shuttle. However, their model is not appropriate for
xylem transport in detopped plants since the water nutrient recycling from shoot to root is interrupted.
One aim of this work was to analyse the concomitant
functioning of the nitrate assimilation pathway and the
exudation process in an attempt to identify the compounds which are involved in the exudation process.
Detopped 7 d seedlings of maize were used as a model.
The effects of NO^~ and of other compounds of the
assimilation pathway, such as NH^, glutamine or asparagine were compared. The specific effect of exogenous
asparagine and glutamine as efficient inducers of exudation could be due either to their nature as end products
of the nitrate pathway or to the fact that they could
release ammonium through deamination of their amide
group. The respective responses of nitrate and ammonium
suppliers on exudation enhancement were investigated.
Another objective was to check that inhibition of the
assimilation pathway really causes a decrease of the
exudation process. Specific inhibitors of nitrate reductase
and glutamine synthetase (GS, EC 6.3.1.2) were used to
limit the assimilation of NO^~ and NH^. Tungstate, a
specific inhibitor of nitrate reductase was used when
KNO3 was supplied in the medium. As a control, tungstate was also given with urea as the NH^ supplier
allowing an inhibition of NR while maintaining a normal
nitrogen assimilation. In this case, any restriction in plant
growth or activity of the major enzymes involved in
malate metabolism and carbon assimilation could be
suspected (Deng et al., 1989a). Two inhibitors of the
glutamine synthetase activity, phosphinothricin (PPT,
Logusch et al., 1991) and methionine sulphoximine
(MSO, Fentem et al., 1983; Lee and Ayling, 1993) were
used. They were compared in two conditions: (i) when
KNO3 was given in the root medium, and (ii) when
NH^ was provided externally through urea acting as
N H / supply. The in vivo functioning of the assimilation pathway was checked from measurements of the
change in sap composition in NO3~, NH4+, and
glutamine + asparagine (Minshall, 1964; Ivanko and
Ingversen, 1971; Oaks and Hirel, 1985). The variations
of solute concentrations confirmed the negative effects of
the inhibitors.
Finally, the initial hypothesis assuming that nitrate
induced enhancement of exudation rate via an increase
of hydraulic conductance rather than of the driving force
for water transport was checked: the large range of
exudation rate of exudation produced by the use of
inhibitors were compared to concomitant measurements
of the total osmolarity of xylem sap.
Materials and methods
Plant material and growth conditions
Seeds of a maize hybrid (Zea mays L., F7F2) were rehydrated
for 24 h in distilled water bubbled with air. One hundred
rehydrated seeds were placed on a germinating bed, the
scutellum facing down, at a density of 12 seeds dm" 2 in a
transparent plastic box (24 x 36 x 13 cm). The germinating bed
consisted of a layer of synthetic porous tissue (1 cm thickness)
covered with three layers of filter paper. Each box contained
500 ml of 2 mM potassium phosphate (pH 7.0). The boxes were
closed and put in a growth chamber maintained at constant
temperature (30 °C) and high relative humidity (95-98%). All
the boxes were connected to an air-flow inlet which provided
water-saturated air at 30 °C. Illumination was provided by a
cool white light source (fluorescent tubes Phillips TL 33 at
200;umol photon m" 2 s" 1 ) 16 h d" 1 .
Experimental protocol for xylem sap collection
Plants were harvested at 7 d, at the end of the light period, 16 h
after the addition of salt and nitrogen compounds in the root
medium. All the plants in a box were rapidly detopped at 2 cm
above the seed. The first drops were discarded to avoid exudate
pollution by damaged cells or phloem sap. Then, the emerging
drops of xylem sap were collected with the aid of a peristaltic
pump. The xylem sap from each box was pooled at 1,3, 5, and
7.5 h. The rate of exudation for 100 plants was routinely
determined and the cumulative volume of exudate calculated.
It may be assumed that the water contained in the sap
originated only through roots because the contact between seed
and tissue and paper bed was negligible.
Salt and nitrogen compounds used as exudation enhancers
Inducers were applied in root medium at day 6, 16 h before
detopping. Each salt or metabolite treatment was given in a
100 ml concentrated solution added to the root medium. As the
initial root medium volume was 500 ml, the solutes were diluted
by a factor of about 6 since the root medium volume could be
slightly modified after 6d of growth. Each box received 100 ml
of a 50 mM salt solution (KC1 or KNO 3 ) providing a
concentration of about 8 mM in the rhizosphere. Urea was
provided at 25 mM, giving a final N H / concentration of 5 mM.
The final concentrations of NO^ and NH44" were specifically
determined as described below for xylem sap samples (data not
shown). For amino acids, the final concentrations were not
checked. Amino acids and urea solutions were supplemented
with KC1 in order to reach the same osmolarity
(100 mOsmol 1 ~') as in the case of KNO 3 and K G applications.
The final osmolarity of the root medium containing inducers
was around 16mOsmol I" 1 in all cases. In an early experiment,
the effect of KNO 3 , K G and H 2 O on exudation was compared
to that of nitrogenous compounds, NH 4 NO 3 , urea [CO(NH 2 ) 2 ]
and glutamine. In another experiment, the effects of selected
amino acids (asparagine, glycine, proline, and phenylalanine)
on exudation were compared to that of H 2 O and glutamine as
control assays.
Reference assays for KN03 and urea treatment
(1) Time-course of KNO 3 and urea effects on exudation. K N 0 3
and urea were applied 2, 6, 12, 26, and 30 h before detopping.
Xylem exudation and nitrate assimilation pathway
(2) KNO 3 and urea as reference assays. These compounds were
applied 16 h before detopping. Exudation rate and solute
concentrations in the sap were followed for 7.5 h. These
reference assays were systematically done with the following set
of inhibitor experiments.
Inhibitor treatments
NR is a homodimeric enzyme carrying a molybdenum cofactor
at the catalytic site (Redinbaugh and Campbell, 1985); tungsten
can substitute molybdenum in the cofactor structure of the NR,
resulting in an inactive enzyme (Heimer et al., 1969). Tungstate
was applied at 0.15 mM as in Deng et al. (19896). As tungstate
is acting through de novo biosynthesis of an inactive protein, a
long-term effect (72 h) was compared to a short-term (16 h)
one. It was added simultaneously with K N 0 3 or urea 16 h prior
detopping in the short-term experiment (ST) or 72 h before
detopping in the long-term experiment (LT). In each case, a
control experiment was performed replacing tungstate by
molybdate (0.15 mM).
GS catalyses the NH^" assimilation into glutamine with
glutamate and ATP as other substrates (Oaks and Hirel, 1985).
As phosphinothricin (PPT) and methionine sulphoximine
(MSO), analogues of glutamate, are acting through competitive
inhibition, the inactivation occurred as soon as the inhibitor
reached the catalytic site. As an example, root GS activity of
barley seedlings was lowered to 20% of the initial value after a
30min incubation with 1 mM MSO (Fentem et al., 1983).
Thus, in the present work, the time schedule was shorter than
in the tungstate experiment: inhibitors were applied at the
onset of detopping for 0 h (initial experiment) or 16 h before
detopping for short-term experiments (16 h). MSO (from
Sigma) and PPT (gift from INRA, Versailles) were used at a
final concentration of 0.25 mM. The purity of the PPT was not
checked. Each analogue was applied together with KNO 3 or
urea treatments at 6 d, 16 h before detopping (ST) or just at
the time of detopping at 7 d on KNO 3 and urea plants (Initial
Term, IT).
Rationale of the method
All the conditions given here was the same as Barthes et al.
(1995a) and, more precisely, there was no calcium added in the
minimum medium. In the preceding paper, the main concern
was the presence of K + in the minimum medium: it modifies
slightly the rate of exudation of the treated plants, but, it did
not change the enhancement due to KNO 3 compared to K G .
In a same way, in separate experiments, it was checked that no
marked effects of Ca 2 + on exudation enhancement due to
KNO 3 compared to K G assays could be observed (Table 1).
Moreover, the further experiments (Barthes et al., 19956)
designed for young wheat seedlings were performed with a
basic medium supplied with Ca2 + : the time-course and the
KNO 3 concentration range which increased exudation rate were
very close to those obtained on maize plants without Ca 2 + . So,
the enhancement of exudation due to KNO 3 is an easily
reproducible phenomenon in various conditions and various
plants as described in Barthes (1994). In the following
study, each comparative result (KNO 3 /KC1, KNO 3 /urea,
inhibited/not) was always obtained in duplicate in a single run
of experiments. This run was repeated twice for each case.
Osmolarity of the collected sap
Osmolarity was determined on 100/xl sap aliquots with an
automatic freezing point depression osmometer (Roebling).
The results were expressed in mOsmoll" 1 . Data lower than
4 mOsmol I" 1 were not significant.
487
Table 1. Exudation rate ofdetopped seedlings growing previously
on a minimum medium supplemented with mono-Ca-phosphate
(+Ca2+) or not (-Ca2+)
Seedlings were detopped after 16 h on water. KC1 or 1CNO3 (lOmM).
Seedlings were grown during the 6 d before detopping in a medium
containing 1.5 mM K-phosphate. 0.5 mM, pH 7 for the Ca 2+ treatment
whereas a medium containing only 2 mM K-phosphate buffer was used
for the treatment without Ca2 + . Results are mean + SEM of n replicates.
Treatment
+ Ca2 +
-Ca
2 +
Exudation rate of seedlings grown on different
media (10"- 1 2 m 3 s - ' )
Water
KC1
KNO 3
5.01+0.35
«=15
5.02 + 0.44
«=12
5.76 + 0.44
«=13
5.60 ±0.48
n=\A
n=\\
A7=ll
12.40 + 0.53
13.20 + 0.09
Carbohydrates and amino acid content in xylem sap
Sap was analysed by an HPLC procedure on 25 pi aliquots at
1, 3, 5, and 7.5 h after detopping, as previously described
(Barthes et al., 1995a). The total concentration for carbohydrates and for amino acids was obtained by adding up the values
of each class of compounds. Results were expressed in mM.
The cumulated amounts of metabolites during the total timecourse of sap collection were calculated using the sap exudate
volume and were expressed in /umol per 100 plants.
Carbohydrates in exudates consisted mainly of glucose and
fructose and a small amount of sucrose. The most abundant
amino acid was glutamine.
Nitrate and ammonium content in xylem sap
Nitrate content was estimated as previously described (Barthes
et al., 1995a) by the nitrite colorimetric method (Aslam et al.,
1976) after an enzymatic conversion of NO^ into NOJ with a
commercial purified nitrate reductase from Aspergillus species
(Sigma N 7265). Free ammonium was estimated by the Nessler
method (Umbreit et al., 1964). The absorbance was determined
on an ELISA microplate on a spectrophotometer (MR 700
Dynatech) at 490 nm. Both results were expressed in mM.
Leaf nitrate reductase activity
At detopping, small leaf pieces were picked up from the median
part of the second leaf until a 1.2 g fraction fresh weight was
collected. Two 1.2 g samples were harvested per box. These
samples were fixed in liquid nitrogen and stored at — 80 °C.
The protocol used to assay in vitro NR activity was adapted
from Aryan et al. (1983) and was previously described (Barthes
et al., 1995a). Results are expressed in pkat g" 1 FW.
Protein content
Leaf soluble protein content was estimated by the Bradford
method (Bradford, 1976) using the Bio-Rad reagent on the
supernatant fluid (lOmin at 4000g) of the extract used for
NR assay.
ELISA test
Leaf NR protein content was estimated by a two-site ELISA
test using a mouse monoclonal antibody, anti-NR ZM 96-9-25,
as coating reagent (Cherel et al., 1986), and a polyclonal
antiserum raised against spinach NR as the second antibody.
Sensitive quantification of NR was conducted using alkaline
phosphatase labelling the enzyme rabbit antibody as described
488
Barthes et al.
by Hoarau et al. (1991). Absorbance values determined at
405 nm in an ELISA spectrophotometer apparatus (MR 700
Dynatech) were linear with extract dilution. A 1/6 dilution was
chosen for comparative ELISA quantification of nitrate reductase protein using nitrate-treated plants as control.
rate and can be regarded as exudation enhancers. Two
families of efficient exudation N-enhancers may be distinguished. The first is composed of nitrate and metabolites
thereof (e.g. ammonium, glutamine) and the second is the
exogenous NH^ donors: urea, NH4NO3 and probably
glutamine and asparagine. Thus, glutamine and asparagResults and discussion
ine are likely to exhibit a dual role, as end-products of
the assimilation pathway and as ammonium donors
Effects of the A/03~ assimilation pathway functioning on the
through the activities of glutamate dehydrogenase or
exudation process
glutaminase from the plant and its rhizosphere (Peeters
N compounds or NH% suppliers: Cumulative amounts of and Van Laere, 1992) and asparaginase (Sieciechowicz
exudate increased with time after detopping showing that
et al., 1989; Peeters and Van Laere, 1992). KNO3 and
maize seedlings were able to maintain the exudation
urea were chosen as reference assays for each family
process during the 5h experimental period (Fig. 1).
because they have a monovalent role, acting either as
Compared to water-plants, exudation was doubled by
endogenous NH^ donor in the case of KN0 3 or as
KC1, but, at the same osmolarity (16mOsmol I" 1 ), mixexogenous NH^ donor in the case of urea. Urea was
tures containing KNO3, urea, NH 4 N0 3 or glutamine
preferred to an ammonium salt because it is well known
induced a stronger enhancement of the exudation (x 2
that NH^1" exhibits some toxicity and generally gives rise
compared to KC1, Fig. 1 left). Asparagine and glutamine to plants with a reduced dry matter content (Salsac et al.,
produced a similar enhancement of exudation whereas
1987). Urea is readily degraded through urease activity
other amino acids tested, glycine, phenylalanine and
from the plant or companion bacteria, thus delaying
proline were less efficient than KC1 (Fig. 1 right). Three
NH^ supply and avoiding the accumulation of large
compounds involved in the nitrate assimilation pathway,
ammonium concentrations. In this way, a decrease of the
", NH.J", glutamine, (asparagine) increased exudation
toxic effect of a high ammonium concentration was
expected.
Comparative study of KN03 and urea supply: Irrespective
of the duration of the treatments before detopping, KN0 3
enhanced exudation more than urea (Fig. 2): exudation
appeared earlier with KN0 3 and was rapidly linear after
2 h of incubation, whereas a pronounced lag phase was
observed with urea for the first 12 h of incubation. It is
likely that the progressive onset of urease activity delayed
the accumulation of NH^. The fact that the lag phase
decreased after longer incubation is consistent with this
hypothesis. A 16 h treatment was chosen for the reference
assay for both inducers because it corresponded to the
o
1
2
3
4
5
0
1
2
3
4
5
Time after detopping (h)
Fig. 1. Effect of different root media on cumulative volume of exudate
collected after seedling detopping. Media were applied 16 h before
detopping. Left, KNO3, urea, NH4NO3, glutamine (GLN) K.C1, and
H2O. Right, glutamine, asparagine (ASN), glycine (GLY), phenylalanine (PHE), proline (PRO), H2O as control. Inducers for both the
experiments were given as 100 ml of solution at 50 mM for KNO3, KC1
and NH4NO3, 25 mM for urea with 17.5 mM of K.C1, and 50 mM for
the different amino acids with 50 mM of KG. As the initial root
volume was 500 ml, the final salt and metabolite concentration was
lowered by a factor 6.
6
12
18
24
30
Incubation duration (h)
Fig. 2. Time-course of KNO3 and urea assays on cumulative exudate
collected after 2, 6, 12, 26, and 30 h.
Xylem exudation and nitrate assimilation pathway
length of the light period. This choice allowed the collection of xylem sap in the dark period and to obtain the
maximum NR activity for KNO3 assay (Barthes et al.,
1995a).
After a 16 h incubation KNO3-treated plants (Fig. 3,
left) had an exudation rate around 1.3 ml h" 1 per 100
plants whereas urea-treated plants (Fig. 3, right) had a
slightly lower value around 1.0 ml h" 1 per 100 plants.
Nitrate is known to be an efficient counterion supporting
potassium uptake and deposition in the xylem (Touraine
and Grignon, 1981) and a greater value of sap osmolarity
could be expected in such conditions, explaining the
higher water flux in KNO3-treated plants. However, this
interpretation is not supported by the sap osmolarity
values (Fig. 4, control plants, open symbols) since the
range of osmolarity of the xylem exudate went from 45
to 35 for the KNO3-treated plants and from 65 to 55 for
TREATMENT
16 h incubation
489
the urea-treated plants. Urea was supplemented with a
KC1 salt in order to maintain the same medium osmotic
condition as in the KNO3 experiment. In such conditions,
urea-treated plants, exhibited the higher osmolarity and
the lower exudation rate. KNO3- and urea-treated plant
osmolarity presented a slight dilution with time as previously described (Barthes et al., 1995a). This fact was
related to the disappearance of the resistance due to the
removal of the vascular system of leaves.
The sap of KNO3 plants contained NO^ and NH4+
(Fig. 3, left) and both presented the time-dependent dilution noted for total osmolarity: the initial NO3~ concentration was 10 mM and declined by 38% in 5 h; the initial
NH^ concentration was lower, 2 mM, but declined more
rapidly, by 55% in 5 h. Exudates of urea plants (Fig. 3,
right) contained twice as much NH^" than the sap of
KNO3 plants but, as expected, no nitrate. Amino
acid concentrations were also higher with urea (3 mM
compared to 1 mM). Similar results were obtained by
Chaillou et al. (1991). The higher sap amino acid concentrations of urea-supplied plants suggests that a large part
of NH^ arising from urea was actually assimilated.
Similar results have already been described by Lee and
Lewis (1994) using either 15N-nitrate or 15N-ammonium.
This interpretation is supported by the fact that carbohydrates decreased in the sap. Carbohydrate concentration
was slightly higher in KNO3 assays at the beginning, but
it dropped more rapidly than in the urea assays. Between
5 and 7 h, carbohydrates totally disappeared from the
sap in both cases, indicating a restriction in their supply.
As the carbohydrate pool was mainly represented by
glucose (70%) and fructose (25%) and slightly by sucrose
(<10%) (Barthes et al., 1995a), it may be assumed that
carbohydrates in the xylem sap were coming from seed
or root metabolism rather than from damaged cells or
phloem sap contamination. The amino acid content in
xylem sap remained almost constant indicating that secretion into the xylem was maintained continuously, in spite
of the detopping. The metabolite composition in the sap
from the KNO3 and urea assays were used now as control
patterns in the following experiments with inhibitors of
the nitrate assimilation pathway.
Action of inhibitors on the NO3 pathway functioning
0 12
3 4 5 6 7
0 1 2 3 4 5 6 7
Time after detopping (h)
Fig. 3. Comparative analysis of the xylem sap collected from detopped
plants exposed to KNO3 (left) and urea (right). Row 1 depicts the
evolution of exudation rate in ml h" 1 100 plants"1, the second row, the
sap NO3" and NFLJ" concentrations (mM), the third row, the sap
carbohydrate and amino acid concentrations (mM). Xylem sap was
collected from 0-1 h, from 1-3 h, from 3-5 h, and from 5-7.5 h.
Inhibition of NR by tungstate: The simultaneous addition
of tungstate and KNO3 in root medium in the short-term
experiments (16 h before detopping) was analysed first.
In order to ascertain if the time of tungstate action was
sufficient to prevent the synthesis of an active NR, it was
decided to use the aerial part of the plants removed at
detopping as an internal control and to measure NR
protein. The total leaf protein amount was constant
whatever the treatment, whereas in vitro NR activity
decreased from 276 to 129 pkat g" 1 FW (Table 2). Thus,
490
Barthes et al.
TUNGSTATE
SHORT TERM
MSO PPT
(i6h)
INITIAL
(O h)
o
E
w
O
E
J5
o
E
o
0
1
2
3
4
5
6
7
8
Time after detopping (h)
2
3
5 6 7
0
1
2
3
4
5
6
7
8
Time after detopping (h)
Fig. 4. Evolution of sap osmolarity of KNO3- and urea-treated plants submitted to tungstate or PPT and MSO inhibition Left, KNO3 plant after
short-term inhibition by tungstate (closed symbols) and control (open symbols). Middle, KNO3 plant after initial inhibition by PPT (closed circles)
and MSO (closed set squares) and control (open symbols). Right, urea plant details as in middle case. Values are means of two independent
experiments.
Table 2. Leaf nitrate reductase activity, leaf NR protein and total
leaf protein content expressed on afresh weight basis from plants
treated (W) or not with tungstate
Exudation effectors were either KNO3 or urea.
Medium
treatment
Leaf protein
content
(mgg- 1 FW)
NR activity
(pkat g" 1 FW)
NR protein
content
(% of control")
KNO 3
KNO 3 + W
Urea
Urea + W
2.56±0.32
2.62 + 0.11
3.51 ±0.12
2.75±0.01
276 + 53 (108)'
129±3 (46)°
112±19(J2)°
42± 13 (15)"
100 + 8
133 + 38
40 + 8
24±3
° In italics NR activity on a total protein basis is indicated.
b
Control is the absorbance value (dilution 1/6) obtained in a linear
portion of the dilution curve for KNO3 control plants (with molybdate
and no tungstate).
the decrease of leaf NR activity was not sufficient to
induce a deficit in leaf protein biosynthesis, which is
consistent with a short-term effect. Surprisingly, NR
protein increased to 130% of the control value. Deng
et al. (19896) obtained a similar result using tungstate on
tobacco for a few days and explained it as a deregulation
of the expression of the nitrate reductase structural gene
(Deng et al., 19896). Compared to KN0 3 plants, urea
plants showed a higher leaf protein content which could
be related to their higher amino acid content in the sap
as previously noted (Fig. 3). A low NR activity was
detected in urea plants (112 pkat g" 1 FW). It fell to 42
pkat g" 1 FW when tungstate was added in the root
medium. Correlatively, the NR protein represented 40%
and 24%, respectively, of the KNO3 plants. This NR
activity in the absence of nitrate in the medium may be
ascribed to a non-inducible NR activity. A constitutive
form was previously described by Oaks et al. (1982) in
maize and was considered as an inactive precursor form.
Remmler and Campbell (1986) also described a constitu-
tive form in maize, but with a low activity. This set of
experiments showed that, in urea plants where the constitutive NR was effective, the NR activity on a protein
basis (Table 2) in tungstate plants decreased by 53%
compared to the non-inhibited plants, together with a
NR protein representing only 60% of the non-inhibited
plants. In contrast, in KNO3 plants, the NR activity on
a protein basis decreased by 55% whereas NR quantity
increased by 33%. This comparison of urea and KNO3
plants suggested that the constitutive NR was less affected
than inducible NR by tungstate. As tungstate acted
through the biosynthesis of an inactive NR, the inducible
NR pool was rapidly modified whereas the constitutive
NR was less affected, especially if its turnover was slow.
As tungstate inhibited the NR activity in the leaf, it was
expected that the whole seedlings were under a tungstate
inhibition status. A root NR study would probably be a
better method of assessing NR inhibition, but it was
preferred to have the sap collection and the inhibition
proof from a single assay. The assumption of root NR
inhibition was supported by the concentrations of some
solutes in the sap as described below.
In the short-term KN0 3 experiment, NH^ and amino
acids were absent in sap from tungstate plants compared
to untreated plants (Fig. 5E, I). By contrast, NO3~ sap
content was slightly higher (Fig. 5E). This result suggests
that the lack of amino acid and ammonium resulting
from NR inhibition enhanced the NO3~~ secretion into the
xylem. This is in agreement with the assumption of a
NH^ or glutamine retroinhibition on NO^ uptake
(Breteler and Siegerist, 1984; Lee et al., 1992; Muller and
Touraine, 1992). Carbohydrates reached a plateau after
3 h and was lower for treated plants (Fig. 51).
Tungstate given together with urea in the root medium
(Fig. 5, ST, urea) did not cause large modifications of the
sap solutes, compared to urea alone: NH^ accumulated
Xylem exudation and nitrate assimilation pathway
491
TUNGSTATE
SHORT TERM
LONG TERM
(i6h)
Urea
KNO3
'
(72 h)
—' c
u
H
K
0 1 2 3 4 5 6 7
0 1 2 3 4 5 6
Time after detopping (h)
2 3 4 5 6 7
0 1 2 3 4 5 6 7
Time after detopping (h)
Fig. 5. Comparative analysis of the xylem sap collected from detopped plants exposed to KNO 3 (left) and urea (right) in the absence (control,
open symbols) or presence (closed symbols) of 0.15 mM of tungstate. The left part (A) gives a short-term action of the inhibitor (16 h) and the
right part (B) gives a long-term action (72 h) Row 1 (A) depicts the evolution of cumulated amount of exudate in ml 100 plants" 1 , the second
row (B), the sap NO3~ and NH^ amounts (^mol 100 plants" 1 ), the third row (C), the sap carbohydrate and amino acid amounts (^mol 100
plants" 1 ). The sap collection was tested at 1,3, 5, and 7.5 h.
regularly to the same extent (Fig. 5F). Carbohydrates
accumulated slightly more in treated than in non-treated
plants where a plateau was reached after 3h (Fig. 5J).
Conversely, amino acids were higher in non-treated
plants. The sum of amino acids plus carbohydrates was
constant in both cases. The nearly normal amino acid
pool in urea-tungstate conditions contrasted with its
absence in KNO3-tungstate plants. Thus, an exogenous
supply of NH^1" could overcome the deficiency of nitrate
reduction as reported by Deng et cil. (1989a).
In long-term experiments, amino acids and NH^" were
nearly absent in the xylem sap for both KN0 3 and urea
plants treated by tungstate (Fig. 5G, H, K, L) as opposed
to short-term experiments (Fig. 5E, F, I, J). Moreover,
xylem NO3" content was accumulated at a lower rate
than control in long-term KNO3-tungstate plants,
whereas it was the reverse in short-term experiments
(Fig. 5E, G). The carbohydrate content of urea-tungstate
plants was severely reduced by a long-term NR inhibition,
but not in short-term conditions (Fig. 5J, L). These facts
could be explained by the side-effects of tungstate in a
long-term action. Other enzymes with molybdenum in
the active site, such as xanthine dehydrogenase, could be
severely disturbed and the general plant metabolism may
be affected. So, in long-term experiments, NR activity
was inhibited in such a way that medium urea could not
supply for the lack of endogenous NH^.
Inhibition of GS by PPT and MSO: The GS inhibition
was conducted either on KNO3 medium providing NH^
endogenously or urea medium providing NH^ exogenously. If both NH^ sources were equivalent substrates
for GS activity, similar responses of the inhibited seedlings
would be expected. The solute content in the sap of
KNO3 plants and urea plants as GS activity was decreased
either by PPT or MSO, is shown in Figs 6 and 7,
492
Barthes et al.
PPT
INITIAL
ShoRT
(Oh)
Urea
Q.
O
O
o
TERM
(i6h)
B
140
^120
1100
I so
O 60
3. 6' 40
Z
20
O B
0
£ a
*
CD
a>
Si
t
20
T3
o
10
£
iS
•
o
i 3
o
<P* 1 2 3 4 5 6 7 ° 1 2 3 4 5 6
0 1 2 3 4 5 6 7
Time after detopping (h)
0 1 2 3 4 5 6 7
Time after detopping (h)
GS
Fig. 6. Comparative analysis of the xylem sap collected from detopped plants exposed to KNO 3 (left) and urea (right) in the absence (control,
open symbols) or presence (closed symbols) of 0.25 mM PPT. The left part gives a short-term action of the inhibitor given at detopping (0 h) and
the right part gives a long-term action (16 h). Row 1 (A) depicts the evolution of cumulated amount of exudate in ml 100 plants" 1 , the second row
(B), the sap NOf and N H / amounts (^imol 100 plants" 1 ), the third row (C), the sap carbohydrate and amino acid amounts (/imol 100 plants" 1 ).
The sap collection was tested at 1,3, 5, and 7.5 h.
respectively. For both inhibitors, sap amino acids
decreased and NH^ increased. The time-courses were
slightly different, MSO being more effective than PPT. In
short-term experiments (16 h), both inhibitors drastically
decreased the sap amino acid content (Fig. 6K, L;
Fig. 7K, L) and increased the sap ammonium content
especially in the case of MSO (Fig. 6G, H; Fig. 7G, H).
MSO restricted amino acid accumulation within 1 h after
application (Fig. 71, J). The PPT effect on amino acid
accumulation was evident within 3 h of application
(Fig. 61, J). Jackson et al. (1993) also noted in maize
that MSO produced an increase of NH^ in root tissue.
To summarize, GS inhibition tends to decrease sap amino
acid content and to increase sap NH^ content. These
results were observed whatever the medium used which
is indicative of GS inhibition. MSO was more efficient
than PPT, but as the purity of the PPT was not available,
it was not possible to ascertain this fact.
Evolution of metabolite contents in sap were in agreement with the fact that inhibition was well established in
short-term treatments for tungstate and initial experiments for PPT and MSO, lateral effects being obvious
for longer durations.
Effects of the assimilation pathway dysfunctioning on the
exudation process
Effect of tungstate on exudation: When the assimilation
pathway was blocked at the NR site in the presence of
KN0 3 , exudation decreased, to a larger extent in longterm inhibition (11 ml per 100 plants to 4 ml per 100
plants, Fig. 5C) than in short-term inhibition (11 ml per
100 plants to 7 m] per 100 plants, Fig. 5A). In this latter
case (short-term inhibition), it is notable (Fig. 4, left) that
the osmolarity of inhibited or control plants varied similarly. Therefore, the differences in flux (Fig. 5A) are
Xylem exudation and nitrate assimilation pathway
MSO
INITIAL
SHORT TERM
(Oh)
493
(i6h)
a>
JO
o
3
E
3
o
0 1
2 3 4 5 6 7
0 1
2 3 4 5 6 7
Time after detopping (h)
o1
GS
2 3 4 5 6 7
0 1 2 3 4 5 6 7
Time after detopping (h)
Fig. 7. Comparative analysis of the xylem sap collected from detopped plants exposed to KNO 3 (left) and urea (right) in the absence (control,
open symbols) or presence (closed symbols) of 0.25 mM MSO. The left part gives a short-term action of the inhibitor at detopping (0 h) and the
right part gives a long-term action ( I 6 h ) Row 1 (A) depicts the evolution of cumulated amount of exudate in ml 100 plants" 1 , the second row
(B), the NO3" and NH4+ amounts (^mol 100 plants" 1 ) in the sap, the third row (C), the carbohydrate and amino acid amounts (^mol 100
plants"'). The sap collection was tested at 1, 3, 5, and 7.5 h.
unlikely to be attributable to an osmotic cause. The
restoration of nitrogen assimilation by exogenous NH^
supply produced two responses depending on the duration
of tungstate treatment. In short-term treatments, the
presence of urea cancelled the tungstate effect on the
exudation (Fig. 5B). In the long-term tungstate treatments, NH4+ supply could not restore exudation (8 ml to
3 ml per 100 plants, Fig. 5D), but the low exudation rate
was associated with a lack of ammonium and amino acid
pools in xylem (Fig. 5H, L). Thus, the relationship
between exudation rate and the presence of the intermediates of the nitrate assimilation pathway remained
valid.
Effect of MSO and PPT on exudation: Despite the slight
difference between MSO and PPT action, the exudation
rate was usually decreased whatever the inhibitors and
the inducer medium (Figs 6, 7). In short-term experiments
(16 h), urea, as expected, did not prevent MSO and PPT
effects: for both media a decrease in exudation rate was
observed (Figs 6C, D; 7C, D). It was more pronounced
in urea-treated plants than in KNO3-treated plants. Upon
initial exposure to either MSO or PPT, exudation rates
of KNO3-treated plants were diminished (Figs 6A, 7A).
However, in urea-treated plants only MSO was effective
(Figs 6B, 7B). In the case of urea-PPT plants, where
exudation did not decrease upon initial exposure to the
inhibitor (Fig. 6B), it should be noted that amino acid
accumulation in the xylem sap was restricted only after
3 h (Fig. 6J). Yet, the decrease of exudation rate by PPT
on urea plants clearly appeared in long-term conditions
(Figs 6D; 7D). In the initial sets of experiments with
KN0 3 and urea (Fig. 4), the change in sap osmolarity
did not follow in any way the corresponding exudation
rate: PPT- and MSO-treated plants on KN0 3 medium
and the control without inhibitor had similar osmolarity
494 Barthes et al.
(Fig. 4, middle) despite a higher water flux for control
plants (Figs 6A, 7A). For urea, osmolarity of control
plants was higher than those of inhibited plants, MSO
having the lower osmolarity (Fig. 4, right); there was
only a decrease in exudation for MSO plants (Fig. 7B),
in contrast with PPT plants which remained unchanged
(Fig. 6B).
To sum up, the dysfunctioning of the assimilation
pathway restricts the enhancement of exudation by
nitrate. Moreover, there was no relationship between sap
flux and osmolarity when NR and GS sites were inhibited.
Therefore, it is assumed that the sap flux could be
modified by the presence of intermediate (s) of the assimilation pathway.
Glutamine role: At this time of this work, the exact role
of glutamine remained unclear in that it can act (i) sensu
stricto as an end-product of the assimilation pathway or
from other metabolic sources or (ii) as an NH^ donor.
It can also be argued that inhibitors, tungstate as PPT or
MSO, may alter the transport of nitrate and ammonium
into the xylem elements and the proportion of the ammonium and amino acids in the xylem sap derived from
nitrate reduction, protein turn-over and endosperm. A
general increase of proteolysis of inhibited plants can be
discarded if it is considered that (i) the sap osmolarity of
inhibited plants was equal to or lower than control plants,
and (ii) the soluble protein content of leaves remained
unchanged. It is known that tungstate is without effect
on NO^ absorption (Heimer and Filner, 1971) and that
GS inhibition increases NH^ absorption (Jackson et al.,
1993; Lee and Ayling, 1993). But the actual origin of the
sap glutamine can not be demonstrated here. However,
the experiments with GS inhibitors (PPT and MSO), in
the presence of urea in root medium, may help discussion
of this point. If glutamine is acting sensu stricto, addition
of urea, together with PPT and MSO, could not restore
exudation. If glutamine was acting through its role of
ammonium supplier, it could be expected that urea would
cancel the GS-inhibitor effect on exudation. In long-term
experiments for both inhibitors and in short-term with
MSO, it is clear that urea can not restore exudation, so
glutamine seems to be directly involved in the exudation
enhancement. The absence of an effect on exudation
during the initial exposure to PPT may simply be due to
a delay in the inhibitor action as discussed above. It could
also suggest that there was different pools of glutamine
having different effects on the exudation process.
the case of the KNO3 application, showed that the
enhancement of exudation was mainly attributable to an
increase in root hydraulic conductance as osmolarity
remained unchanged. The present work reinforces this
lack of relationship between osmolarity and flux in the
inhibitory experiments. There is a more convincing relationship between flux and the presence of products of the
assimilation pathway, supporting the assumption that the
assimilation chain could modify water conductance. These
relationships were noted in a positive way as intermediates
of the assimilation pathway increase water flux. They
were also seen in a negative way as the dysfunctioning of
the pathway is accompanied by a decrease of the flux.
Thus, a question remained open: what is the relationship
between the NO3~ reduction metabolic pathway in roots
and the root conductance to water? Such a question may
be resolved if the synthesis of the recently described
proteins involved in water transport (Chrispeels and
Maurel, 1994) is triggered by the NO3~ assimilation
pathway.
Conclusion
The present results support the specific effect of
N-compounds on exudation rate and specify the effect
linked to the assimilation pathway. They clearly demonstrate that the functioning of the nitrate assimilation
pathway enhances the exudation process whereas its
inhibition decreases the exudation process. Furthermore,
exudation was maintained despite NR inhibition when a
proper feeding of the amino acid pool was obtained by
supplying ammonium through urea. The simultaneous
inhibition of the first step of the nitrate assimilation
pathway and addition of the reduced product of the
pathway showed that intermediates of the nitrate assimilation pathway are more likely to be directly involved in
the enhancement of exudation than NOf itself. Further
investigations, using inhibitors of GS activity (MSO and
PPT) in the presence of KNO3 resulted in an inhibition
of exudation. The fact that an exogenous supply of
glutamine stimulated exudation tends to suggest that the
nitrate and ammonium effects on exudation could be
mediated by the glutamine production. The comparison
of sap osmolarity and exudation rate also argues for an
action of nitrate (and other compounds of the assimilation
chain) on root water conductance as previously assumed
(Barthes et al., 1995a, b).
Nitrate assimilation pathway and root water conductance:
Nitrate metabolism interferes with the water flux through
the root (Jv). Jv is controlled by two parameters: the
Acknowledgement
osmotic potential difference between external medium
and xylem (A *¥) and the hydraulic conductivity of the
We thank Dr M Caboche for his gift of anti-NR mouse
pathway Lp (Boyer, 1985). Barthes et al. (1995a, b) in
monoclonal antibody, anti-NR ZM 96-9-25.
Xylem exudation and nitrate assimilation pathway
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