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2004
27810351046
Original Article
Putative role of g -aminobutyric acid (GABA) as a longdistance signal in up-regulation of nitrate uptake in Brassica
napus L.
N. BEUVE1,*, N. RISPAIL2,*, P. LAINE1, J.-B. CLIQUET1, A. OURRY1 & E. LE DEUNFF1
1
ABSTRACT
The relationship between nitrate influx, BnNrt2 nitrate
transporter gene expression and amino acid composition of
phloem exudate was investigated during N-deprivation
(short-term experiment) and over a growth cycle (longterm experiment) in Brassica napus L. The data showed a
positive correlation between g-aminobutyric acid (GABA)
in phloem exudate and nitrate uptake in the short- and the
long-term experiments. The hypothesis that this non-protein amino acid could up-regulate nitrate uptake via a longdistance signalling pathway was tested by providing an
exogenous GABA supply to the roots. The effect of GABA
was compared with the effects of Gln, Glu and Asn, each
known to be inhibitors of nitrate uptake. The results
showed that GABA treatment induced a significant
increase of BnNrt2 mRNA expression, but had less effect
on nitrate influx. By contrast, Gln, Glu and Asn significantly reduced nitrate influx and BnNrt2 mRNA expression compared with the control plants. This study provides
the first evidence that GABA may act as a putative
long-distance inter-organ signal molecule in plants in
conjunction with negative control exerted by Gln. The upregulation effect of GABA on nitrate uptake is discussed
in the context of its role in N metabolism, nutritional stress
and the recent discovery of a putative role of GABA as a
signal molecule in plant development.
Key-words : Brassica napus L.; amino acids; BnNrt2 genes;
g-aminobutyric acid (GABA); high-affinity transport system (HATS); nitrate uptake; phloem; translocation.
INTRODUCTION
The high-affinity transport system (HATS) is one of the two
classes of transport systems involved in nitrate uptake in
higher plants (Forde 2000). It operates from low external
Correspondence: Erwan Le Deunff. Fax: +33 0231 565360; e-mail:
ledeunff@ibfa.unicaen.fr
*These authors contributed equally to this work.
2004 Blackwell Publishing Ltd
Experimental treatments
Experiment 1 (N-deprivation)
Two sets of plants were transferred to N-free nutrient
solution for different durations (0, 12, 24, 48, 72 h). At
each sampling time, HATS activity and BnNrt2 mRNA
abundance were measured in one set of plants and amino
acids content in phloem exudates was assayed with the
other set.
2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 10351046
lection period of 12 h. The EDTA of each collected fraction was precipitated by adding 100 mL of 0.5 N HCl
and stored for 1 h at -20 C. The samples were then
stored over night at 4 C prior to centrifugation at
5000 g for 10 min. The supernatants were adjusted to
pH 5.9, filtered through a 0.45 mm nylon membrane and
stored at -20 C before amino acid analyses. Free amino
acids in phloem exudates were analysed by high-performance liquid chromatography (HPLC) as ophtaldialdehyde derivatives on a C-18 column using a 32 Karat
System (Beckman Instruments, San Ramon, CA, USA)
as previously described by Murray, Hatch & Cliquet
(1996). Specific amino acids were quantified using aaminobutyric acid as an internal standard.
2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 10351046
RESULTS
15
NO3 influx and BnNrt2 mRNA level during Ndeprivation and over the growth cycle
When plants previously fed with NO3 were transferred to
N-free solution, the 15NO3 influx kinetic showed a transient
increase (about 1.4-fold) during the first 24 h (Fig. 1a) and
then a decrease from 24 to 72 h of N-deprivation. Similarly
BnNrt2 mRNA level increased by 2.5 fold after the first
24 h of N-deprivation and decreased thereafter to a value
lower than the control plants (Fig. 1a & c).
The 15NO3 influx increased during the bolting period
of the growth cycle, followed by a drastic decline at the
flowering stage and a slight increase during pod filling.
The abundance of BnNrt2 mRNA mirrored the evolution of 15NO3 influx, except at stage E where no BnNrt2
mRNA was detected, suggesting that a specific regulation might exist at the transcriptional level around the
flowering period when remobilization of N occurs in the
plants.
(a)
(c)
(b)
(d)
Figure 1. Changes in 15NO3 influx and BnNrt2 gene expression during the time-course of N-deprivation and over the growth cycle in
Brassica napus L. plants. Plants were grown directly in hydroponic conditions for 2 months (N-deprivation) or were acclimated at C2 stage
from the field (growth cycle) in a nutrient solution with 1 mM nitrate. For N-deprivation, plants were transferred to a solution without nitrate
(time t = 0). In these two experiments, influx rate was measured at 100 mM with K15NO3. Values are the means of three replicates of plants
(N-deprivation) or nine replicates (growth cycle). The vertical bars on nitrate influx values indicate SD for n = 3 and SE for n = 9 when
larger than the symbol. For BnNrt2 mRNA relative expression, vertical bars indicate SD for n = 2 when larger than the symbol.
2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 10351046
Figure 2. Evolution of total amino acid content in phloem exudates during the time-course of N-deprivation and over the growth cycle in
Brassica napus L. plants. Total amino acid content was determined in phloem exudates collected between 8 and 10 h and 1012 h of exudation
for N-deprivation and growth cycle experiments, respectively. Values are the means of six plants. All vertical bars indicate SE (n = 6) when
larger than the symbol.
2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 10351046
Figure 3. Evolution of individual amino acids present in phloem exudates of Brassica napus L. plants during the time course of Ndeprivation and over the growth cycle. Values are the means of six plants. All vertical bars indicate SE for n = 6 when larger than the symbol.
2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 10351046
Figure 4. Correlations between variations of nitrate influx and contribution of GABA or Gln in phloem exudates during N-deprivation
and over the growth cycle in Brassica napus L. plants. All vertical bars indicate SE for n = 6 (GABA or Gln) when larger than the symbol.
All horizontal bars indicate SD for n = 3 (N-deprivation influx) and SE for n = 9 (growth cycle influx) when larger than the symbol.
2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 10351046
(a)
(b)
(c)
DISCUSSION
Relationship between glutamine in phloem
exudate and nitrate uptake during N-deprivation
and over the growth cycle
It is well established that N-deprivation leads to a transient
de-repression of high-affinity nitrate influx and Nrt2 gene
expression in plants with a low N status (Lejay et al. 1999;
Forde 2002). In plants with a high N status, de-repression
seems to be bypassed, leading to a direct decline of nitrate
uptake interpreted as de-induction (Clarkson 1986; Siddiqi et al. 1989; Faure-Rabasse et al. 2002). Our results for
15
NO3 influx and BnNrt2 gene expression (Fig. 1a & c) are
consistent with these previous studies.
This de-repression is classically interpreted as a depletion of the internal amino acid pool through continued
translocation, assimilation and protein synthesis (Cooper &
Clarkson 1989; Lejay et al. 1999). In our N-deprivation
experiment, the increase in total amino acid measured in
the phloem exudate (Fig. 2a) contradicts this assumption.
However, we observed that the relative contribution of Gln
was inversely related to both nitrate uptake and BnNrt2
expression level although no significant correlation was
found (r2 = 0.753, P > 0.05; Fig. 4c). This lack of significant
correlation with Gln can be attributed to the low number
of kinetic data points (n = 5). Indeed, negative effect of Gln
have been clearly established on gene expression and
nitrate uptake in many species by exogenous supply to the
root (Vidmar et al. 2000). Our results in Fig. 5 also confirmed the role of Gln as potential negative effector
although no correlation was found after exogenous amino
acid supply to the root between variations of nitrate influx
and the root concentration of Gln or other amino acids
(Table 1).
In consequence, our results support partially the hypothesis of Vidmar et al. (2000) that Gln could be the main
down-regulator of nitrate uptake and a putative longdistance inter-organ signal in planta (Nazoa et al. 2003).
However, three main results of the present growth cycle
experiment provide evidence which contradicts the hypothesis of N uptake down-regulation by total amino acids or
Gln content: (i) the high content of total amino acids
recorded in phloem exudates during the bolting period
when N uptake increases (Figs 1b & 2b), which is not consistent with a main role of amino acids in down-regulation
of nitrate uptake; (ii) the lack of nitrate uptake de-repression at the beginning of pod filling (G2 stage) when total
amino acid content in the phloem exudate was at its lowest
value (Figs 1b & 2b); (iii) the lack of a significant correlation between 15NO3 influx and relative Gln contribution in
the phloem exudate (r2 = 0.44, P > 0.05; Fig. 4d). These
results must, however, be interpreted with caution. The
dual role of some amino acids, both as nutrients and as
signals, and their possible interconversion or compartmentalization in root tissues is potentially problematic in
phloem sap analysis when attempting to correlate influx
variations with amino acid composition.
In long-term experiments and non-limiting N conditions,
signals other than amino acids may be involved, as suggested by the lack of BnNrt2 mRNA transcripts at stage E
(preflowering stage) when nitrate uptake was at its highest
level. Similar result has been previously described by
Nazoa et al. (2003) in Arabidopsis around the flowering
period where the expression of GUS gene driven by the
AtNrt2.1 promoter give a lack of signal in the root. Likewise, Vidmar et al. (2000) reported that after 24 h of nitrate
induction on N-deprived plants, HvNrt2 expression
decrease to undetectable levels while nitrate influx
2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 10351046
Control
100 mM GABA
1 mM GABA
1 mM Gln
1 mM Glu
1 mM Asn
Asp
(nmol g-1 FW)
Glu
(nmol g-1 FW)
Asn
(nmol g-1 FW)
Gln
(nmol g-1 FW)
GABA
(nmol g-1 FW)
370 139
247 43
344 63
379 65
314 80
369 74
876 295
694 90
1038 305
1266 277
887 155
850 192
327 123
164 40
231 110
323 116
143 28
1684 515
506 168
460 126
413 167
753 277
341 60
718 240
494 251
308 85
1105 237
369 76
361 39
323 130
Values shown are the means of three independent replicates SD of the mean.
remained around its maximum level. In this context, Rossato et al. (2002) proposed that the post-flowering decline
of N uptake could be a result of the production of methyl
jasmonate (a growth regulator) by young growing tissues
or senescing leaves during the flowering period. Foliar
application or supply of methyl jasmonate to roots during
the vegetative period induced early leaf senescence and was
associated with a drastic decrease of NO3 uptake (Rossato
et al. 2002). Alternatively, Malagoli et al. (2004) suggested
that modification of carbon partitioning around the flowering period could favour newly appearing sink tissues to the
detriment of the C allocated to the root. This last hypothesis
implies the regulation of N uptake by C status, as demonstrated by the partial restoration of Nrt2.1 gene expression
level and NO3 influx after exogenous supply of sucrose to
the root at night (Lejay et al. 1999).
2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 10351046
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Received 19 December 2003; received in revised form 1 April 2004;
accepted for publication 13 April 2004
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