Download Are phloem amino acids involved in the shoot to root control of NO

Journal of Experimental Botany, Vol. 49, No. 325, pp. 1371–1379, August 1998
Are phloem amino acids involved in the shoot to root
control of NO− uptake in Ricinus communis plants?
3
Pascal Tillard, Lucien Passama and Alain Gojon1
Biochimie et Physiologie Moléculaire des Plantes, ENSA-M/INRA/UM2/CNRS URA 2133, Place Viala,
F-34060 Montpellier Cedex 1, France
Received 5 January 1998; Accepted 14 April 1998
Abstract
The putative role of phloem amino acids as negative feedback signals for root NO− uptake was investigated
3
in Ricinus communis L. The NO−-grown plants were
3
subjected to N-deficiency due either to complete
N-deprivation, or to localized N-deprivation on one side
of a split-root system. In comparison with controls, complete N-deprivation resulted in a transient increase in
15NO− influx, and in profound changes in downward
3
phloem transport of amino acids. Total amino acid concentration in the phloem sap decreased by 40%, but
responses markedly differed between the individual
amino acids. Concentrations of Gln and Ser were rapidly
lowered by 50%, while those of Val, Phe, Leu, and Ile
displayed a marked increase. Localized N-deprivation on
one side of the split root system also resulted in the
up-regulation of 15NO− influx in the roots still supplied
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with NO−. However, the amino acid composition of the
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phloem sap directed to these roots was not modified by
the treatment, and remained similar to that in N-sufficient
control plants. Only amino acid transport to the
N-deprived roots was affected as observed in response
to complete N-deprivation. The results from split-root
plants indicate that the response of root NO− influx to
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N-deficiency is controlled by shoot-borne regulatory signals, and provide a case study where these signals are
not related to a qualitative change or a significant
decrease in downward phloem transport of amino acids.
Key words: N-deficiency, NO− uptake, phloem amino
3
acids, Ricinus communis, whole plant signalling.
Introduction
Considerable evidence indicates that the root uptake of
inorganic ions is regulated in order to match the require-
ments for growth and development (Glass, 1983;
Clarkson, 1988; Rodgers and Barneix, 1988; Grignon,
1990; Imsande and Touraine, 1994). This seems to result
from changes in the expression of the various root uptake
systems, which are specifically down-regulated under high
nutritional status of the plant for the corresponding ion
or element (Clarkson and Lüttge, 1991; Logan et al.,
1997). Little is known about the endogenous signals
triggering this response, but a common hypothesis is that
root uptake systems are under negative feedback control
by the root concentration of the ion they take up (or of
its metabolic products) (Glass, 1983, 1988; Clarkson,
1988; Grignon, 1990; Lee et al., 1992). However, this is
not sufficient to account for all aspects of the control of
root ion uptake. Indeed, numerous studies have demonstrated that the regulatory mechanisms are not located in
the root only, but integrated at the whole plant level
(Clarkson, 1988; Pitman, 1988; Grignon, 1990; Imsande
and Touraine, 1994). For instance, experiments with splitroot plants showed that NO− uptake by roots well
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supplied with NO− is de-repressed in response to
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N-starvation experienced by the other portion of the splitroot system (Drew and Saker, 1975; Burns, 1991; Lainé
et al., 1995). This stresses the point that root NO− uptake
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is ultimately regulated by signals transported from the
shoot to the roots (Clarkson, 1988; Grignon, 1990;
Imsande and Touraine, 1994).
Two series of observations support the idea that amino
acids are the main phloem-borne signals for feedback
regulation of NO− uptake (Imsande and Touraine, 1994).
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First, ample cycling of amino acids occurs between the
shoot and the roots (Simpson et al., 1982; Gojon et al.,
1986; Cooper and Clarkson, 1989; Marschner et al.,
1996), which provides the basis for a mechanism integrating the N status of the whole plant and conveying this
information to the roots. Second, increases in downward
1 To whom correspondence should be addressed. Fax: +33 4 67 52 57 37. E-mail: [email protected]
© Oxford University Press 1998
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Tillard et al.
phloem transport of several amino acids result in an
inhibition of NO− uptake similar to that observed after
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exogenous supply of these compounds to the roots
(Muller and Touraine, 1992). The regulatory model postulates that cycling of amino acids to the roots (i.e. the
transport root  shoot  root) is inhibited by low N
status of the plant, or high N demand for shoot growth.
The corresponding decrease in amino acid delivery to the
roots is thought to explain the well-known up-regulation
of NO− uptake associated with N-deficiency (Lee, 1993),
3
or increase in growth rate (Rodgers and Barneix, 1988).
Although tempting from a theoretical viewpoint, the
model is still controversial (Lainé et al., 1995), mainly
because of the limited information available on the control
of the amino acid cycling between the shoot and the root.
In conflict with the model, several reports suggest that N
deficiency results in an increase, rather than in a decrease,
of amino acid cycling in the shoot (Lambers et al., 1982;
Rufty et al., 1984; Duarte and Larsson, 1993; Peuke et al.,
1994; Marschner et al., 1996). However, these data generally refer to total amino-N, and do not provide detailed
information on a putative modification of the amino acid
spectrum of the phloem sap in response to N-deficiency.
Since the various amino acids differ markedly in their
inhibitory effect on NO− uptake (Breteler and Arnozis,
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1985; Muller and Touraine, 1992), this leaves the possibility that shoot-to-root signalling of N status may be more
related to qualitative, rather than to quantitative changes
in downward phloem transport of amino acids.
The aims of this study were to investigate the changes
in the phloem concentrations of the various amino acids
in response to N-deficiency, and tentatively to relate these
changes to the regulation of 15NO− influx in the roots.
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Several experiments were carried out with NO−-grown
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Ricinus communis plants, submitted either to complete
N-deprivation, or to localized N-deprivation on a portion
of a split-root system. The regulation of 15NO− influx
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in the NO−-fed roots of the plants with localized
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N-deprivation is truly indicative of remote control mediated by phloem-borne signals. Thus, a crucial point in
the identification of these signals was to set up a protocol
for specifically collecting the phloem sap directed to
these roots.
Materials and methods
Plant culture
The seeds of Ricinus communis L. (cv. Palma Christi) were
disinfected for 15 min in sodium hypochlorite, thoroughly
rinsed, and sown on perlite watered with tap water. After 3–4 d
in the dark, the germinated seedlings were transferred to 10 l
plastic containers (6–8 seedlings per container) filled with a
complete nutrient solution containing: 1 mM KNO , 0.5 mM
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Ca(NO ) , 1 mM KH PO , 1 mM MgSO , 0.1 mM FeNa3 2
2 4
4
EDTA, 50 mM KCl, 30 mM H BO , 5 mM MnSO , 1 mM ZnSO ,
3 3
4
4
1 mM CuSO , and 0.1 mM (NH ) Mo O . The plants were
4
4 6
7 24
then grown until the age of 17–21 d, depending on the
experiments, in a growth chamber under the following conditions: constant relative humidity at 70%, 14/10 h light/dark
cycle, 25/20 °C light/dark temperature, and a light intensity
(PPFD) of 400 mmol m−2 s−1. During the growth period, the
nutrient solutions were renewed three times per week to avoid
depletion.
Experimental conditions
Three types of experimental set-ups were used to investigate the
response to N-deficiency (Fig. 1). The ‘whole’ plant set-up
(Fig. 1A) refers to intact plants left on the 10 l containers, with
the whole root system exposed to a unique nutrient solution.
The ‘split root’ set-up (Fig. 1B) refers to plants with the root
system separated in two unequal parts, each one located in a
separate container. The installation of the split root plants was
carried out the day before the beginning of the experiments. A
few roots (10–20% of the root system) were selected and placed
in a 0.6 l container, while the remaining major part of the root
system was placed in an adjacent 2.5 l container. Both containers
were filled with fresh complete nutrient solution. The ‘splitstem’ set-up (Fig. 1C ) is identical to the ‘split-root’ set-up,
except that the separation of the two sides of the root system
was carried out by prior surgical treatment. For this purpose,
the tip of the main root of the 3–4-d-old seedlings was cut, and
the basal part of this root, together with the few basal cm of
the hypocotyl, were carefully separated longitudinally using a
razor blade. A piece of parafilm was inserted between the two
parts of the hypocotyl to prevent healing. The seedlings were
then grown under the same conditions as the intact ones. At
the time of the experiments, these plants had the base of their
stem separated into two parts, each supporting approximately
half of the root system. They were treated the same way as
‘split-root’ plants, i.e. transferred the day before any treatment,
with each part of the root system in a separate container filled
with complete nutrient solution.
Experiments of N, S or P starvation on ‘whole plants’ were
carried out by substituting the complete nutrient solution by
minus-NO−, minus-SO2− or minus-Pi ones, respectively. These
3
4
anions were replaced by Cl− in such a way that the
concentrations of the various cations were not modified. Control
plants were simultaneously given fresh complete nutrient
solution. Experiments of localized N-deprivation on either
‘split-root’ or ‘split-stem’ plants were carried out by substituting
the complete nutrient solution feeding one part of the root
system (the major part for ‘split-root’ plants) by the minusNO− solution. The other part of the root system received fresh
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complete nutrient solution. The roots directly experiencing
N-deprivation were called ‘treated roots’, while those remaining
on the complete nutrient solution were called ‘untreated roots’
(Fig. 1). Control plants were given the complete nutrient
solution to both sides of the root system. Starting from the day
before the experiments, all solutions were at pH 5.8, and were
renewed every day.
Influx of 15NO− was determined by total 15N incorporation
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in the tissues, as described in Delhon et al. (1995). For ‘whole’
plants, 15NO− influx was assayed on the whole root system
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after transfer of the plants for 1 min in 0.1 mM CaSO , then
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for 5 min in complete nutrient solution containing 0.2 mM
K15NO (99.9 atom% 15N, pH 5.8), and finally for 1 min in
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0.1 mM CaSO . The influx solution included CaSO and K SO
4
4
2 4
to kept Ca2+ and K+ concentrations identical to those in the
nutrient solution used for growth of the plants. For ‘split-root’
and ‘split-stem’ plants, 15NO− influx was assayed on the
3
untreated roots only. The protocol was the same as for ‘whole’
Regulation of NO− uptake 1373
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Fig. 1. Principle of the experimental designs used in the experiments. ‘Whole’ plants (A) refer to plants for which the root system has been
uniformly supplied either with the complete nutrient solution (control ), or with the minus-NO− solution (N-deprived ). ‘Split-root’ plants (B) refer
3
to plants with the root system separated in two unequal parts placed in separate containers. ‘Split-stem’ plants (C ) refer to plants submitted to a
surgical separation of the stem base in two parts, with the corresponding roots placed in separate containers. Control ‘split-root’ or ‘split-stem’
plants were supplied with the complete nutrient solution on both sides of the root system. N-deprived ‘split-root’ or ‘split-stem’ plants were supplied
with the complete nutrient solution on one side of the root system ( Untreated roots), and with the minus-NO− solution on the other side
3
( Treated roots).
plants, except that the plant was not transferred, but the roots
were carefully lifted out of their container to allow its
replacement by another one containing the proper solution to
be used (CaSO or 15NO−). At the end of the final rinse in
4
3
CaSO , roots were separated from shoots, and treated roots
4
were separated from untreated roots, where appropriate. The
organs were then weighed and dried for 48 h at 70 °C prior to
15N determination.
Phloem exudates were collected from shallow incisions made
on the stem, using a razor blade (Hall et al., 1971). To
investigate the phloem sap moving predominantly downward
to the root system, all incisions were made below the
cotyledonary node. In ‘whole’ and ‘split-root’ plants, the
incisions were made at random locations on the stem. In ‘splitstem’ plants, the incisions were made on each stem base, and
the exudates bleeding from either stem base were collected
separately. Unless otherwise stated, the exudate drops were
collected for 120 min in a capillary tube, and stored in preweighed Eppendorf tubes maintained on ice (1 tube per plant
for ‘whole’ and ‘split-root’ plants, 1 tube for each side of the
stem base of ‘split-stem’ plants). The amount of phloem exudate
was determined gravimetrically, and the tubes were stored at
−20 °C until amino acid analysis.
For determination of amino acid concentrations in the tissues,
the organs were separated after harvest of the plant, weighed,
and stored in liquid N .
2
Analytical protocols
Total N and 15N contents of the tissues were assayed mass
spectrometrically, using the ANCA-MS system ( Europa
Scientific Ltd, Crewe, UK ), on 2–3 mg of dried ground samples
(Clarkson et al., 1996).
The amino acids were extracted from the frozen tissues by
grinding in 96% ethanol, followed by four successive hydro-
alcoholic extractions (96%, 80%, 60% ethanol, and deionized
water) at 4 °C, according to the procedure described previously
(Delhon et al., 1995).
The measurement of amino acid concentrations in both plant
extracts and diluted phloem exudates (dilution 1/100 in
deionized water) was done by HPLC, according to Muller and
Touraine (1992). This technique, involving fluorimetric determination of o-phthaldialdehyde-derivatives of amino acids,
allowed a highly sensitive and accurate assay of concentrations
of most amino acids, except Pro, Cys and His. Trp and Lys
concentrations were found to be negligible (Jeschke et al.,
1997), and were not routinely assayed.
Results
Effect of N-deprivation on root 15NO− influx and downward
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phloem transport of amino acids in ‘whole’ plants
Transfer of the ‘whole’ plants to the minus-NO− solution
3
resulted in a fast and dramatic decrease of the total N
content of the roots, from 5.50±0.28% dry weight at the
time of the transfer to 3.72±0.37% and 2.10±0.15% dry
weight 24 h and 96 h after the transfer, respectively. In
comparison with control plants, a transient stimulation of
root 15NO− influx was observed in the N-deprived plants,
3
24 h after the transfer to the minus-NO−solution (Fig. 2).
3
Thereafter, 15NO− influx in N-deprived plants declined
3
and fell below that measured in control plants (Fig. 2).
The analysis of the amino acid composition of the
phloem sap showed important changes in response to
N-deprivation ( Fig. 3). The total amino acid concentra-
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Tillard et al.
Fig. 2. Effect of transfer of the ‘whole’ plants to a minus-NO− solution
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(N-deprived plants) on root 15NO− nflux measured at 0.2 mM 15NO−.
3
3
The results are expressed as a percentage of the root 15NO− influx in
3
control plants. The initial value of 15NO− influx was
3
80.9±32.6 mmol h−1 g−1 DW. The results are the means of six
replicates ±SE.
tion ranged from 110 to 150 mM in the exudates of
control plants during the time-course of the experiment,
but was decreased by 40% in the exudates of N-deprived
plants 24 h after the transfer to the minus-NO− solution.
3
This percentage was not markedly modified later on, and
total amino acid concentration in the phloem sap of
N-deprived plants remained approximately half that of
control plants. However, besides the overall decrease in
total amino acid concentration, there were major differences in the response of individual phloem amino acids
to N-deprivation (Fig. 3). Three classes of amino acids
could be distinguished. The first class included Gln and
Ser ( Fig. 3), and also Glu, Asp, Asn, and Gly (not
shown), for which a significant decrease in concentration
was observed in N-deprived plants as compared to control
plants. This decrease was particularly pronounced for
Gln and Ser, the two main amino acids of the phloem
sap, and could be observed as soon as 12 h after transfer
to the minus-NO− solution (data not shown). The second
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class corresponded to the amino acids the concentrations
of which were not significantly modified by N-deprivation,
and comprised Thr ( Fig. 3), and also Ala, Arg, Tyr, and
Met (not shown). The third class is represented by minor
amino acids like Val and Leu ( Fig. 3), and also Phe and
Ile (not shown), the concentrations of which were initially
not affected by N-deprivation, but increased markedly
(up to a factor of 10 for Leu) after 48 h. The above
changes in the phloem concentrations of amino acids
appeared quite specific for N-deprivation, and were not
observed in plants deprived of Pi or SO2− for 72 h
4
( Fig. 4).
Effect of localized N-deprivation on root 15NO− influx and
3
downward phloem transport of amino acids in split-root
plants
Fig. 3. Time-course of amino acid concentrations in the phloem
exudates collected from control (open symbols) or N-deprived (closed
symbols) ‘whole’ plants. The phloem exudates were collected for
120 min from shallow incisions on the stem below the cotyledonary
node, at various times after the transfer of the N-deprived plants to the
minus-NO− solution. The results are the means of six replicates ±SE.
3
To investigate specifically the shoot-to-root signalling
involved in the control of root NO− uptake, two inde3
pendent experiments were carried out using ‘split-root’
plants (Fig. 1B). In these plants, N-deficiency was initiated by exposing the major part of the root system to the
minus-NO− solution (treated roots), while NO− was
3
3
supplied to a few roots only (untreated roots).
The localized N-deprivation resulted in a decrease of
total N and NO− concentrations in both treated roots
3
and shoots, but did not affect those in the untreated roots
left on the NO− solution (data not shown). In response
3
to the N-deprivation experienced by the treated roots, 15
NO− influx was stimulated in the untreated roots of
3
‘split-root’ plants ( Fig. 5). This stimulation was of the
same magnitude as that previously measured on the
‘whole’ plants (Fig. 2). Localized N-deprivation also led
to changes in phloem amino acid concentrations ( Table 1)
very similar to those previously observed in response to
N-deprivation of ‘whole’ plants ( Fig. 3). The concentra-
Regulation of NO− uptake 1375
3
Table 1. Effect of localized N-deprivation on amino acid concentrations in the phloem sap of ‘split-root’ plants
The plants were either supplied with complete nutrient solution on both
sides of the root system (control ), or were subjected to localized
N-deprivation by supplying the treated roots with a minus-NO−
3
solution for 48 h (see Fig. 1). The results are the means of six
replicates ±SE.
Fig. 4. Effect of N, P or S-deprivation on the amino acid concentrations
in the phloem exudates of ‘whole’ plants. Control plants were left on
the complete nutrient solution, while N, P or S-deprived plants were
transferred 72 h prior to the exudate collection to minus-NO−, minus3
Pi, or minus-SO2− solutions, respectively. The results are the means of
4
six replicates ±SE.
Fig. 5. Response of 15NO− influx in the untreated roots of ‘split-root’
3
plants to the transfer of the treated roots to a minus-NO− solution.
3
The results are expressed as a percentage of the 15NO− influx measured
3
in the corresponding side of the root system of control plants (see
Fig. 1 for details of the experimental protocol ). The initial value of
15NO− influx was 131.1±30.6 mmol h−1 g−1 DW. The results are the
3
means of six replicates ±SE.
Amino acid
Control plants
(mM )
N-deprived plants
(mM )
Gln
Ser
Glu
Asp
Ala
Thr
Val
Gly
Asn
Tyr
Arg
Phe
Ile
Leu
Total
63.5±5.6
23.6±4.5
21.7±2.6
15.6±1.7
8.37±0.59
3.65±0.77
1.70±0.35
1.13±0.12
0.83±0.24
0.67±0.05
0.42±0.19
0.23±0.10
0.25±0.08
0.25±0.08
142.8±11.8
36.9±10.9
14.7±4.1
19.3±2.3
13.4±3.1
8.05±1.11
3.57±0.39
2.27±0.47
0.88±0.21
0.45±0.10
0.33±0.31
0.42±0.12
0.77±0.26
0.83±0.24
0.52±0.24
103.2±20.3
tions of Gln, Ser and Asn were 40% lower in N-deprived
plants than in control plants, while those of Val, Phe, Ile,
and Leu were 50–200% higher. Surprisingly, a marked
increase in the flow rate of phloem exudation was
observed in response to localized N-deprivation ( Fig. 6),
but this increase was significant only 48 h after the
beginning of the treatment. Accordingly, the exudation
rates of Gln and Ser, calculated by multiplying their
concentrations in the phloem by the flow rate of exudation, were only reduced during the first 48 h after the
transfer of the treated roots to the minus-NO− solution,
3
but not after (data not shown).
The total free amino acid concentration in treated roots
and shoots was lower in N-deprived plants than in control
plants ( Fig. 7). These changes were limited to the four
Fig. 6. Cumulative exudation of phloem sap collected either on control
(open symbols) or N-deprived (closed symbols) ‘split-root’ plants. The
N-deprived plants were supplied with the minus-NO− solution on the
3
treated roots 48 h prior to the collection of the phloem exudates.
The results are the means of six replicates ±SE.
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Tillard et al.
Fig. 7. Concentrations of free amino acids in the organs of ‘split-root’
plants 48 h after the transfer of the treated roots of N-deprived plants
to the minus-NO− solution. Control plants were supplied with complete
3
nutrient solution on both sides of the root system (see Fig. 1 for details
of the experimental protocol ). The results are the means of five
replicates ±SE.
major amino acids, Gln, Ser, Glu (Fig. 7), and Asp (not
shown). However, the N-deprivation experienced by the
treated roots had no effect on the concentrations of any
of the amino acids in the untreated roots still supplied
with NO− (Fig. 7 and other data not shown).
3
The effects of 48 h localized N-deprivation on 15NO−
3
influx in the untreated roots, and amino acid concentrations in the phloem sap and in the various organs were
fully reversed by re-supply of 2 mM NO− for 24 h to
3
both sides of the root system (data not shown).
Differential phloem transport of amino acids to NO−3
supplied or N-deprived roots within the same plant
One uncertainty related to the use of ‘split-root’ plants is
that the phloem exudates, collected at random locations
on the stem, were probably a mix of the two saps directed
to treated or untreated roots, respectively. Thus, it is not
known whether the changes in the phloem amino acids
observed in response to N-deprivation ( Table 1) are truly
representative of what occurs for shoot-to-root transport
of these compounds to the untreated roots where
15NO− influx is upregulated (Fig. 5). The ‘split-stem’
3
plants ( Fig. 1C ) were used to solve this problem. The
fact that the stem bases supporting the two sides of the
root system were separated by prior surgical treatment
allowed the specific collection of the phloem saps feeding
treated and untreated roots, respectively. Three independent experiments were done using ‘split-stem’ plants, which
gave similar results.
As in ‘split-root’ plants, supplying a minus-NO− solu3
tion to the treated roots of ‘split-stem’ plants did not
modify the total N concentration in the untreated roots,
but resulted in a significant stimulation of their 15NO−
3
influx (+36% and +90% after 24 h and 48 h, respectively,
data not shown). In control plants, the amino acid
compositions of the phloem exudates collected on either
sides of the stem base were identical (data not shown).
In N-deprived plants, however, the exudates collected on
treated or untreated sides (i.e. on the stem bases supporting treated and untreated roots, respectively) clearly
differed in their amino acid concentrations 24 h (not
shown) and 48 h after the beginning of the treatment
( Table 2). In the treated side, the amino acid concentrations were modified by N-deprivation as described
previously for ‘split-root’ and ‘whole’ plants. The concentrations of Gln, Ser, Glu, Asp, and Asn were lower than
in the controls, while those of Val, Phe, Ile, and Leu were
higher. These modifications were never observed in the
exudates collected on the untreated side, in which the
Table 2. Comparison of amino acid concentrations in the phloem
saps feeding either NO−-fed or N-deprived side of the root system
3
of ‘split-stem’ plants
In control plants, the results presented are the means of the
concentrations measured in all exudates. The N-deprived plants were
subjected to localized N-deprivation for 48 h. The phloem sap was
collected separately on the two stem bases supporting treated or
untreated roots, respectively (Fig. 1). The results are the means of six
replicates ±SE.
Amino acid
Gln
Ser
Glu
Asp
Ala
Thr
Val
Gly
Asn
Tyr
Arg
Phe
Ile
Leu
Total
Control plants
(mM )
41.1±1.9
15.3±2.4
21.8±1.7
16.5±1.2
2.78±0.49
2.45±0.54
1.58±0.47
2.17±0.59
1.00±0.14
2.00±0.49
0.17±0.08
0.18±0.10
0.27±0.10
0.18±0.10
107.3±5.7
N-deprived plants (mM )
Untreated roots
(+NO−)
3
Treated roots
(−NO−)
3
50.1±9.0
10.6±2.4
22.8±4.2
16.4±1.4
3.57±0.52
2.55±0.55
2.10±0.48
2.28±0.66
0.87±0.14
2.20±0.21
0.33±0.05
0.22±0.08
0.35±0.10
0.23±0.10
114.7±12.1
28.0±6.6
7.78±2.1
17.5±3.5
13.5±1.3
2.75±0.50
2.63±0.45
2.27±0.26
2.05±0.73
0.62±0.15
2.17±0.33
0.38±0.08
0.60±0.25
0.92±0.22
0.65±0.18
82.0±11.4
amino acid concentrations remained closely similar to
those measured in the exudates of control plants
( Table 2). After 48 h of treatment, phloem exudation
rates from treated and untreated sides of N-starved plants
were 50% and 90% higher than those measured in control
plants, respectively (data not shown).
Discussion
The up-regulation of 15NO− influx in Ricinus roots in
3
response to N-deprivation of the plant (Figs 2, 5) is a
classical feature interpreted as a relief of the repression
of NO− transport systems by nitrogen metabolites (Glass,
3
1988; Burns, 1991; Clarkson and Luttge, 1991; Lee et al.,
1992; Lee, 1993; Lainé et al., 1995). In roots of N-deprived
‘whole’ plants, the stimulation of 15NO− influx is transient
3
( Fig. 2), which is explained by the opposite effects of
both initial de-repression due to shortage of N metabolites, and subsequent de-induction due to lack of
NO− (Clarkson, 1986). The untreated (NO−-supplied)
3
3
roots of N-deprived ‘split-root’ plants did not suffer
N-deficiency, as indicated by their unaffected total N,
NO−, and free amino acid concentrations ( Fig. 7).
3
However, they reacted to the N-deprivation experienced
by the treated roots, and displayed the classical stimulation of 15NO− influx (Fig. 5). This clearly shows that the
3
shoot-borne signals of N-deficiency overrode the root
signals of N-sufficiency, to result in the compensatory
up-regulation of NO− uptake.
3
In spite of some variation probably due to differences
in the age of the plants or in environmental conditions
between independent experiments, the phloem sap concentrations of the various amino acids in control plants
( Fig. 3; Tables 1, 2) are almost identical to those reported
by Jeschke et al. (1997), with the four main amino acids
Gln, Ser, Glu, and Asp representing up to 90% of the
total amino acid concentration. When looking at the bulk
phloem sap collected from ‘whole’ and ‘split-root’ plants,
two important changes in the amino acid spectrum were
associated with N-deficiency ( Fig. 3; Tables 1, 2). First,
a pronounced decrease in the concentrations of Gln and
Ser occurred within the first 24 h after transfer to the
N-free solution. Second, there were delayed but large
increases in the concentrations of Val, Ile, Leu, and Phe.
The decline in phloem concentrations of Gln and Ser was
associated with similar changes in the leaf concentrations
of these amino acids ( Fig. 7), probably related to shortage
of NO− assimilation in N-deprived plants. The signific3
ance of the increases in the phloem concentrations of the
hydrophobic amino acids Val, Leu, Ile, and Phe remains
unknown. It is not surprising to find a common response
of the three branched chain amino acids, i.e. Val, Leu
and Ile, since their biosynthetic pathways are considered
to be parallel and subject to common regulation through
the activity of acetolactate synthetase (ALS, EC 4.1.3.18,
Regulation of NO− uptake 1377
3
Bryan, 1990). However, the limited knowledge of the
factors affecting ALS (Coruzzi, 1991) prevents any proposal about the relationship between the activity of this
enzyme and the N status of the plant.
A major finding from our experiments with ‘split-stem’
plants with a localized N-deprivation is that the above
changes in amino acid concentrations in response to
N-deficiency were specifically observed in the phloem
sap feeding the treated roots directly experiencing
N-deprivation (Table 2). The phloem sap directed to
untreated (NO−-supplied) roots remained unaffected in
3
comparison with control N-sufficient plants. These results
indicate that a selective partitioning of the N-assimilates
between the various parts of the root system may not
rely only on a difference in sink strength, but can also be
due to unequal phloem delivery arising in the shoot. In
which way the shoot preferentially directs phloem constituents to certain roots is unclear. This may indicate
specific connections between particular leaves and roots.
However, the occurrence of both large root/shoot cycling
and xylem/phloem exchanges of amino acids (Cooper and
Clarkson, 1989; Da Silva and Shelp, 1990; Marschner
et al., 1996) does not favour the idea of separate circuits
for amino acid transport between particular shoot and
root parts.
The changes in the absolute rates of shoot-to-root
transport of amino acids in reponse to N-deprivation are
difficult to assess from these results. The total amino acid
concentration in the phloem sap of N-deprived plants
was 40–50% lower than in controls ( Fig. 3; Table 1).
However, this was apparently compensated after 48 h of
N-deprivation by an increase in the phloem sap flow in
both ‘whole’ plants (data not shown) and ‘split-root’
plants (Fig. 6). More direct quantitative determinations
of shoot-to-root N transport also agree with the idea that
N-limitation does not always result in a decreased export
or cycling of amino acids to the roots (Lambers et al.,
1982; Rufty et al., 1984; Duarte and Larsson, 1993; Peuke
et al., 1994; Marschner et al., 1996). In the N-deprived
‘split-stem’ plants, phloem exudation rates were increased
on both treated and untreated sides in comparison with
controls, but the decrease in the phloem amino acid
concentrations occurred in the treated side only (Table 2).
Thus, the portion of the root system still fed with NO −
3
apparently had preferential (see above) and also increased
phloem import of amino acids in response to
N-deprivation of the other side of the root system. This
conclusion may appear surprising, but is supported by
previous studies showing that in split-root plants with
uneven NO− supply, shoot to root N transport to each
3
subroot correlates with the level of its external NO−
3
supply (Agrell et al., 1994).
Most of the above considerations do not agree with
the hypothesis of a predominant role of phloem transport
of amino acids in the shoot-to-root control of NO−
3
1378
Tillard et al.
uptake. Clearly, the observation that in plants with localized N-deprivation, the phloem amino acids are preferentially directed to the NO−-fed roots (Table 2) displaying
3
the up-regulation response of 15NO− influx (Fig. 5) is
3
not consistent with the idea that these amino acids are
negative feedback effectors of NO− uptake systems.
3
Moreover, up-regulation of 15NO− influx in these roots
3
occurred without any qualitative or quantitative change
in the tissular levels of free amino acids (Fig. 7; see also
Lainé et al., 1995). From a theoretical viewpoint, assuming that phloem amino acids repress the expression or
activity of NO− transport systems implies a dual role for
3
these compounds, both as nutrients and as signals. The
localized N-deprivation (which also corresponds to localized NO− supply) is a physiological situation where these
3
two roles may contradict each other. It has been known
for years that localized NO− supply results in a specific
3
stimulation of the growth of the NO−-fed roots (Drew
3
and Saker, 1975; Granato and Raper, 1989). An important part of the amino acids available to sustain this
increased growth does not come from local NO− assimila3
tion, but derives from phloem import (Lambers et al.,
1982; Agrell et al., 1994). Thus, the increased and preferential phloem transport of all amino acids to the NO−3
fed side of the root system, suggested both by our results
and by previous studies (Agrell et al., 1994), may correspond to a required change in the partitioning of the N
assimilates to meet the local N-demand for increased
growth (Marschner et al., 1996). However, this is not
consistent with the hypothesis of a signalling role of
phloem amino acids, which necessitates a specifically
lowered transport of one or several of these compounds
to the NO−-fed roots displaying the up-regulation
3
response for 15NO− influx. One possibilty to reconcile
3
the opposite interpretations on the role of phloem amino
acids in the regulation of NO− uptake would be to
3
consider that these compounds are not the primary signals
responsible for shoot-to-root control of NO− uptake
3
systems, and that most, but not all, conditions leading to
a change in their cycling to the roots also result in a
change in the downward transport of yet unknown signalling molecules for the regulation of NO− uptake.
3
Acknowledgements
The authors wish to thank Fanny Vanel for excellent technical
assitance, Dr JK Vessey for helpful discussion on the manuscript,
and Professor DT Clarkson for his kind supply of the protocol
used to obtain ‘split-stem’ plants.
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