Journal of Experimental Botany, Vol. 43, No. 310, pp. 1079-1085, May 1997
Journal of
Experimental
Botany
Influence of BAP and NAA on the expression of nitrate
reductase in excised chicory roots
Christophe Vuylsteker, Olivier Leleu and Serge Rambour1
Laboratoire de Physiologie et Genetique Moleculaire Vdgetales, Universite des Sciences et Technologies de
Lille, F-59655 Villeneuve d'Ascq Cedex, France
Received 30 September 1996; Accepted 15 January 1997
Abstract
Introduction
In young planttets of chicory (Cichorium intybus L. var
Witloof) nitrate reduction is mainly localized in roots.
Following root excision, nitrate reductase activity
rapidly decreased. This inhibition was first induced by
phosphorylation of NR, followed by the decrease of
NR-protein and NR-mRNA contents (Vuylsteker et al.,
1997). Addition of 1 0 " M BAP re-induced NRA in
excised roots after 2 d, in vivo NRA reaching 10-fold
the level of controls on day 5. This increase of NRA
was related to enhanced NR-protein and NR-mRNA
contents. After 4 d, BAP increased 16-fold the
NR-protein and 3.5-fold the NR-mRNA levels, respectively. In vitro NRA measured in the presence of EDTA
was 5.6-fold higher in BAP-treated roots than in control roots confirming the in vivo measurements. The
high discrepancy between the NR-protein level and
NRA shows that limiting factors other than the
NR-protein content affected NRA reactivation. With
1 0 " M NAA, in vivo NRA was enhanced seven times
on day 5 and in vitro NRA was increased only 2.5 times.
Enhancement of the NR-protein was more restricted
with NAA than with BAP. Besides a poor effect at the
transcriptional level, NAA may control NRA by a phosphorylation mechanism underscored by higher sensitivity of in vitro NRA to magnesium. Resumption of NRA
in the presence of either BAP or NAA occurred simultaneously to the increase of the dry weight and was
probably driven by increasing needs of reduced
nitrogen to support regrowth.
Plants usually reduce nitrate in their leaves where energy
and reducing power arise (Beevers and Hageman, 1980).
In chicory, a biennial Asteraceae, nitrate reduction occurs
mainly in roots until they differentiate tubers. Thereafter,
nitrate reductase activity is localized in leaves (Dorchies
and Rambour, 1985). Roots of young chicory plantlets
behave as sinks for carbon and sources for nitrogen.
Nitrate reduction in roots depends on the import of
photosynthates which provide carbon skeletons, energy
and reductants (Huppe and Turpin, 1994). In plants
which reduce nitrate in their roots, tight correlations
between root and shoot metabolism are important.
Nitrate reductase, a key enzyme in the control of
nitrogen assimilation, is the target of several regulatory
processes. The availability of nitrogen and carbon particularly affects nitrate reductase which is inducible by nitrate,
whereas ammonium and amino acids can inhibit its
activity (Solomonson and Barber, 1990; Li et al, 1995;
Sivansankar and Oaks, 1995). Carbon metabolism also
regulates nitrate reductase and, in leaves, the light effect
on transcription of NR-mRNA can be mimicked by
sucrose (Cheng et al., 1992; Vincentz et al., 1993).
Moreover, light controls nitrate reductase activity in
leaves by phosphorylation-dephosphorylation reactions
(Glaab and Kaiser, 1995; Huber et al., 1992; Kojima
et al., 1995). In roots, light modulates nitrate reductase
probably indirectly by photoassimilates (Merlo et al.,
1994). These regulatory processes involving both carbon
and nitrogen metabolism, assign to nitrate reductase an
essential function in regulating the carbon: nitrogen ratio
and shoot:root allocation (Beck, 1994).
Cytokinins are increasingly considered as potential
messengers of the nitrogen status towards the root to
Key words: Chicory, BAP, NAA, roots, nitrate reductase.
1
To whom correspondence should be addressed. Fax: + 33 3 20 43 68 49. E-mail: ramboureuniv-lille1.fr
Abbreviations: ATPase, ATP synthase; BAP, 6-benzyl aminopurine; GS, glutamine synthetase; NAA, naphthalene acetic acid; NR, nitrate reductase;
NRA, nitrate reductase activity; TUB, tubulin.
© Oxford University Press 1997
1080
Vuylsteker et al.
shoot allocation of biomass (Beck, 1994). Concentration
of cytokinins within roots and xylem responds to the
nitrogen availability in terms of either total nitrate availability (Samuelson and Larsson, 1993) or of differential
distribution of nitrate along the lateral roots (Samuelson
et al., 1995). Their transport is submitted to fluctuations
of evapotranspiration and hence to foliar activity (Beck,
1994). They are known to regulate NR activity in response
to light or nitrate induction (Lu et al., 1990, 1992;
Samuelson et al., 1995). In Agrostemma githago embryos,
cytokinins induced NRA only in the presence of ethylene
(Schmerder and Borris, 1986).
Among numerous effects on growth and plant development, auxins stimulate root initiation and the development of lateral roots. Data concerning relationships
between auxins and nitrate reductase are, until now, rare
and conflicting (Knypl, 1979). However, a relationship
between the development of NRA and the rhizogenic
potential of IAA and IBA on rooting of pea cuttings has
once been reported (Ahmad, 1988).
When roots of young chicory plantlets were excised
and tranferred to liquid medium, nitrate reductase activity
rapidly decreased via a phosphorylation mechanism.
Thereafter both the NR-protein and NR-mRNA levels
decreased. This inhibition was considered as an adaptative
reduction of the nitrogen assimilatory pathway to a
senescence-like process, as a result of the suppression of
the shoot to root correlations (Vuylsteker et al., 1997).
In sucrose-starved roots, nitrogen assimilation decreased
at the expense of reallocation of reduced nitrogen
(Brouquisse et al., 1991).
As low concentrations of cytokinins and auxins have
long been considered to delay senescence (Goldthwaite,
1987), the possible effects of both growth factors on the
level of nitrate reductase in excised roots were investigated. Moreover, both hormones are implicated in the
control of rhizogenesis and are stored or synthesized in
roots (Torrey, 1976), so they could influence NRA in
chicory roots. NRA, NR and NR-mRNA levels were
measured in BAP- and NAA-treated excised roots with
the purpose of studying their potential reactivating effects.
Materials and methods
Plant material
Chicory seeds {Cichorium intybus L. var. Witloof, cv. Flash)
were surface-stenlized and germinated on solid growth medium
H15, containing 15 mM sucrose, salts of Heller (1953) and
7 g I" 1 agar. The growth chamber was maintained at 22± 1 °C
with a photoperiod of 16/8 h (light/dark) and a light irradiance
of 1 4 f i M m " 2 s " ' . After 18 d, plants which developed two
cotyledons and four leaves were decapitated, and 12 uniform
roots were transferred, in aseptic conditions, into flasks
containing 50ml HI5 liquid medium. NAA and BAP at a
final concentration of 10" 6 M, were aseptically added after
autoclaving the medium, using 0.20 ^un Acrodiscs®.
In vivo nitrate reductase activity
The roots were harvested at different times, weighed and
assayed for NRA according to the in vivo method of Jaworski
(1971). Individual roots were introduced in 2 ml of the
incubation mixture comprising 62.5 mM KN0 3 , 37.5 mM
K-phosphate buffer pH 7.5 and 1.2% 1-propanol (v/v).
Measurements were made on five independent samples and
repeated at least three times. The reaction tubes were submitted
to a flow of nitrogen for 30 s, stoppered, and incubated for
20 min in the dark at 28 °C. Nitrite was revealed by adding
0.5 ml sulphanilamide (11 mM in 3 M HC1) and 0.5 ml of
aqueous 10 mM N-\ naphthyl ethylene diamine dichloride.
NRA was expressed as nmol nitrite produced min" 1 g" 1 FW.
In vitro nitrate reductase activity
In vitro assays are derived from Merlo et al. (1995). Roots were
frozen and ground in a chilled mortar. Extraction buffer
contained 50 mM HEPES-KOH pH 7.5, 5 mM MgCl2, 0.5 mM
EDTA, 14 mM 2-mercaptoethanol, 0.1% (v/v) Triton X100,
10% (v/v) glycerol, 50 ^M leupeptin, 0.5 mM PMSF, and 10%
(w/v) polyvinylpyrrolidone. Extracts were then desalted on to a
G25 Sephadex column equilibrated with the same buffer except
that it contained neither EDTA nor MgCl2. NRA was assayed
using 50 mM, pH 7.5 HEPES-KOH buffer comprising 10 mM
K.NO3, 0.2 mM NADH, and 10 MM FAD.
Modulation of the activation status of NR in vitro, was
performed by adding either 2 mM EDTA or 5 mM MgCl2 to
desalted extracts. Incubation was performed at 30 °C for 5 min,
and the reaction was then stopped by adding 50 /xl 0.5 M zinc
acetate. Excess NADH was oxidized with phenazine methosulphate (final concentration 10fiM). Nitrite was revealed as
above and NRA activity was expressed as nmol of
nitrite min" 1 mg" 1 protein. The protein content was measured
according to Bradford (1976) with bovine serum albumin as a
standard.
ELISA immunoquantification of NR proteins
The NR level was quantified by the two sites ELISA procedure
according to Cherel et al. (1986) using monoclonal anti NR
maize 96925 and S6 polyclonal anti NR maize antibodies. These
antibodies were first tested against chicory root NR by Western
blot analysis and immunoprecipitation assays.
Total RNA extraction
Total RNA was extracted from the root tissues according to a
procedure derived from Chirgwin et al. (1979). One gram tissue
was ground in liquid nitrogen to a fine powder which was
suspended in 5 vols of 4 M guanidium thiocyanate containing
0.1 M TRIS-HC1 (pH7.5) and 1% (v/v) 2-mercaptoethanol.
Nucleic acids were then extracted by phenol/chloroform coupled
with ethanol precipitation (0.75 vol. ethanol and 0.08 vol. 1 M
acetic acid). Nucleic acids were pelleted and dissolved in 10 mM
TRIS-HC1 pH 7.5. RNAs were selectively precipitated with 2 M
lithium chloride. RNA were finally dissolved in diethyl pyrocarbonate treated sterile water.
Northern analysis
20 ng total RNA were run in a 1.5% (w/v) agarose formaldehyde
gel (Sambrook et al., 1989). Subsequently, blotting was achieved
on Hybond-N + (Amersham) membranes. DNA probes were
labelled with [a- 32 P]dCTP (111 T Bq mM " ' ICN) using random
priming (T 7 Quickprime Pharmacia). Hybridizations were
performed according to Church and Gilbert (1984); membranes
were then exposed to X-ray films (Kodak X-Omat AR) at
Hormonal control of nitrate reductase activity
-80°C using intensifying screens. Intensity of the bands was
estimated after scanning and digitalization using a Microtek
Color/Gray scanner (Biorad) connected to a Macintosh LCIII
(Apple) computer. Trie software used was the free ware
NIH-1.56.
The probes were: NR, a partial cDNA from nitrate reductase
of Cichorium intybus (X 84102 EMBL Data Library; Palms
et a]., 1996); GS1, a complete cDNA of cytosolic glutamine
synthetase of Nicotiana tabacum (gift of B Hirel, unpublished
data); ATPase, a cDNA of a /3 subunit of Nicotiana plumbaginifolia (Boutry and Chua, 1985); TUB, cDNA from a tubulin of
Daucus carota (Borkid and Sung, 1985).
Results
In vivo NRA
NRA in roots which were excised from 18-d-old plantlets,
and transferred into liquid medium rapidly decreased
(Vuylsteker et ai, 1997). Addition of either BAP or NAA
in the liquid medium enhanced significantly in vivo NRA
in detopped roots, 48 h after their transfer (Fig. 1). This
effect, estimated by the ratio between in vivo NRA in
roots treated with a growth factor and NRA in excised
control roots, was dependent on the concentration of
both the auxin and the cytokinin. The higher ratios were
obtained by adding 10~6 M BAP or 10"6 M NAA (data
not shown).
In the presence of 10 ~6 M BAP, the values of the NRA
ratios reached 3 at the 2nd and 10 at the 5th day of the
culture (Fig. 1). On day 5, NRA recovered the level of
NRA in intact roots. Thus BAP exerted a long-term
effect, resulting in the recovery of NRA measured in
undetopped roots. However, BAP was unable to prevent
the early phosphorylation reaction which occurred imme-
1081
diately after excision and which induced a rapid inhibition
of NRA (Vuylsteker et ai, 1997). After 6d without
renewing the medium, roots got brown and NRA finally
dropped, reaching the level of NRA in detopped control
roots. Nutritional starvation linked to BAP physiological
effects on roots or to hormonal exhaustion can be inferred.
In the presence of 10~6 M NAA, the NRA ratio
between NAA-treated roots and control roots reached a
value of 7 on day 5 and subsequently declined from the
6th day on (Fig. 1). However, the decrease of NRA after
the 5th day was less important in roots grown with 10~6
M NAA, than in roots grown with 10 ~6 M BAP.
Moreover, stimulation of NRA by 10~6 M NAA was of
less importance than the enhancement induced by 10 ~6
M BAP.
Growth
Dry weight (DW) of control roots remained stable during
the course of the culture (Fig. 2). The loss of nitrate
reduction in excised roots was thus partly correlated to
the absence of growth and subsequent reduced needs of
amino acids. Addition of BAP or NAA to the liquid
medium increased DW of roots from, respectively, the
2nd and the 3rd day on (Fig. 2). After 5 d, DW of BAPor NAA-treated roots were 4-fold higher and 2-fold
higher than the DW of control roots, respectively. The
increase of DW occurred simultaneously with enhanced
NRA (Fig. 1).
In vitro NRA
NRA was shown to be controlled by reversible phosphorylation, the phospho-NR binding stoichiometrically
BAP / Control
NAA/Control
1
24
48
72
96
120
Time (hours)
Time (hours)
Fig. 1. Time-course of in vivo NRA in excised roots grown with 10~6
M of either BAP or NAA. In vivo NRA of excised roots was measured
at different times after 10~6 M of either BAP or NAA were added.
Enzymatic activities are expressed as: NRA in hormonal conditions/
control NRA. Means±SD (n=\0).
Fig. 2. Time-course of dry weight of chicory roots in control conditions
and in the presence of 10~6M of either BAP or NAA. Dry weight was
measured on samples consisting of 12 roots. Means of three repeats are
shown ± S D .
1082
Vuylsteker et al.
to an inhibitor protein. According to these data, models
for reversible control of NRA involving interaction of
phosphorylated NR and nitrate reductase inhibitor
proteins were hypothesized (Kaiser and Huber, 1994;
MacKintosh et al., 1995; Bachmann et al., 1995).
Addition of EDTA to the reaction mixture inhibits the
binding reaction and, consequently, the bulk of NR
protein, phosphorylated or not, contributes to NRA.
Conversely, magnesium stabilizes the inactive phosphoNR-NIP complex (MacKintosh et al., 1995). Thus, the
ratio between NRA measured in the presence of Mg2 +
ions and NRA measured in the presence of EDTA
accounts for the activation rate of NR and reflects the
ratio of active NR-protein.
When in vitro NRA was first measured in the presence
of either EDTA or Mg 2+ ions, in roots harvested on
18-d-old plantlets, the activation rate reached 40%. In
excised roots grown for 3 h in the control medium, the
activation rate reached less than 15%, indicating that
excision induced an enhancement of inactive NR. As the
assays were carried out without adding any protein phosphatase inhibitors, the level of inactive NR may be
underrated. However, as the procedure was carried out
rapidly, this may be insignificant. When the growth time
lasted for 5 d, the activation rate was not measurable,
since NRA in the presence of Mg 2+ ions was not detectable. In excised roots of chicory grown for 5 d in the
presence of 10" 6 M of either BAP or NAA, in vitro NR
activities measured in the presence of either EDTA or
Mg 2+ ions were stimulated. On day 5, NRA assayed with
EDTA was 5.6 times higher in BAP-treated roots than in
controls. In the presence of NAA, NRA was increased
only 2.5 times. Assayed with Mg 2+ ions in vitro NR
activities were 4.3 and 2.9 times higher in roots grown
with BAP and NAA, respectively. Activation rates
reached 34% and 49% in roots grown with BAP and
NAA, respectively (Table 1).
NR-protein
The level of total soluble proteins per gram fresh weight
was not significantly affected by NAA and was enhanced
by only 20% in the presence of BAP, after 5 d (data
not shown).
The level of NR-protein in roots grown with 10~6 M
BAP, rapidly exceeded the level of NR-protein in roots
of undetopped plantlets. It increased linearly reaching a
maximum on day 4, when it was 1 ? times higher than in
controls; on day 4, it remained 13 times as high as in
controls and subsequently decreased (Fig. 3).
In roots grown with 10~6 M NAA the NR-protein
level increased only from the 3rd day on. Compared to
control excised roots, the NR-protein level was enhanced
4 times on day 4, 7.5 times on day 5 and 13 times on day
6 (Fig. 3).
Comparing in vitro NR activities measured with EDTA
and the levels of NR-protein showed discrepancies
between NR activities and the NR-protein levels. The
increment of the NR-protein levels induced by both the
growth factors not only preceded increased NRA, but
equally exceeded the increment of NRA. For instance in
roots grown with BAP, in vitro NRA measured with
EDTA was 5.6 as high as in controls on day 5, whereas
the NR-protein level was 13 times higher. Similar discrepancies between the NR-protein level and NRA occurred
in roots grown with NAA; whereas the NR-protein level
was 7 times higher than in controls, in vitro NRA was
only 2.5 times higher on day 5. Thus in both cases, the
NR-protein level did not match NR activities measured
in vitro, indicating that part of NR was inactive.
400-
300-
Table 1. In vitro NR activities with either EDTA or Mg*+ ions
NR activities were first measured in intact roots. Some roots were then
excised and transferred into liquid media containing 10~ 6 M of either
BAP or NAA; NRA was assayed either 3 h or 5 d after the transfer.
The ratio: Mg^ + NRA/EDTA NRA x 100, represents the activation
rate due to dephosphorylation. NRA values were expressed as nmol
NOf nun" 1 mg" 1 total soluble proteins (means±SD for three determinations) and were typical of data obtained in three different
experiments.
Before
excision
In vitro NRA
with EDTA
In vitro NRA
with MgCl 2
Activation rate (%)
12±1.6
4.9 ±1.4
40
Control
10"*M NAA
10"«M BAP
200-
I
100-
After excision
3h
5d
Control
Control
10.8±1.2 3.4± 1.6
nd
nd
BAP
NAA
19±1.3 8.7±0.7
6.5±1.6 4.3± 1.2
34
49
Time (hours)
Fig. 3. Time-course of the NR-protein level in excised roots grown in
the presence of 10~6 M of either BAP or NAA. NR-protein levels in
excised roots were quantified by ELISA. NAA and BAP were added
aseptically to roots 3 h after they were detopped and transferred into
liquid medium. One significant experiment among three repeats is shown.
Hormonal control of nitrate reductase activity
Northern blot analysis
The mRNA levels of nitrate reductase (NR) cytosolic
glutamine synthase (GS1) and /3-ATPase decreased in
excised control roots indicating that overall transcriptional activity probably decreased in the absence of any
growth factor (Vuylsteker et al., 1997).
Conversely, in roots grown with 10~6 M BAP, high
levels of NR, GS1 and /3-ATPase mRNAs were maintained through the time-course of the experiment (Fig. 4).
This was confirmed by scanning the hybridization bands
(Table 2). However, the level of these mRNAs declined
in control roots grown without BAP, and as a result BAP
reinduced transcription of the NR, GS1 and /3-ATPase
genes. Four days after the transfer in liquid medium
containing BAP, compared to control roots of day 4, the
levels of NR, GSland /9-ATPase mRNAs were increased
3.5, 8.5 and 11.5 times, respectively. The increment of
NR-mRNA occurred simultaneously with the increment
of the NR-protein level (Fig. 3).
Similarly, when excised roots were transferred into
NAA-containing media, no significant decrease of the
NR-mRNA levels was observed (Table 2). Conversely,
Day 4
Day 3
C
B
N
C
B
N
NR
ATP synthase
TUB
GS1
Fig. 4. Northern blot analysis of total RNA of excised chicory roots
grown for 3 and 4 days in the absence or the presence of 10~6 M BAP
or NAA The time scale corresponds to different times of the culture.
C, control; N, NAA; B, BAP. Probes were cDNAs of nitrate reductase
(NR), /3-subunit of ATP synthase (ATP synthase), a-tubulin (TUB),
cytosolic glutamine synthetase (GS1).
Table 2. Densitometric quantification of mRNAs
Fig. 4
evidenced in
Data are given in arbitrary units. Intensity of the different bands of the
controls on day 3 were arbitrary set at 100. As the signal with the
tubulin probe was very weak, an accurate determination of the tubulin
expression was prevented.
3d
4d
Probes
Control
BAP
NAA
Control
BAP
NAA
NR
ATP synthase
GS1
888
140
260
180
120
210
220
40
20
20
140
230
170
90
40
50
1083
between days 3 and 4, the /3-ATPase and GS1 mRNAs
levels decreased in NAA treated roots as in control roots.
However, on day 4, the levels of GS1 and /3-ATPasemRNAs remained 2-fold higher in NAA-treated roots
than in roots grown without NAA.
Discussion
Excision of the roots of young plantlets of chicory and
their transfer in a stirred liquid medium, induced a rapid
loss of NRA, which was due to phosphorylation of NR.
This resembled senescence (Vuylsteker et al., 1997).
Indeed, no significant growth was detected in excised
roots. Similarly, in maize, nitrate reduction decreased in
senescing detached leaves, in sucrose-starved roots or
after decreasing light intensity delivered to maize plantlets
(Brouquisse et al., 1991; Merlo et al., 1994; Saglio and
Pradet, 1980).
Supplying excised chicory roots with either 10~6 M
BAP or 10"6 M NAA restored in vivo NRA, 2 d after
they were transferred. Resumption of NRA was related
to the induction of growth in roots grown with either
BAP or NAA. The inference was that the NRA increase
was driven by needs of nitrogen essential for growth
resumption. In vitro NRA increased as well, but to a
lesser extent. A similar discrepancy between in vivo and
in vitro NRA has already been reported (Oaks, 1992).
Moreover, in vitro NR activities did not match the
NR-protein levels in chicory roots, grown with either
BAP or NAA. In vitro NRA depends on the NR-protein
content, which seldom limits nitrate reduction. Thus,
Arabidopsis thaliana mutants impaired in the expression
of NR and fed with nitrate as the sole source of nitrogen
only retained 10% of in vitro NRA of the wild type and
grew normally (Wilkinson and Crawford, 1993).
Upon the addition of BAP, the level of NR-protein
increased and declined concurrent with the level of
NR-mRNA, both reaching a peak on day 4. NRA
remained unchanged during the course of the first day of
the culture, and subsequently increased reaching a maximum on day 5. On day 4, the levels of NR-mRNA and
NR-protein were 3.5 and 15 times as high as in controls,
respectively. Thus BAP induced enhanced transcription
of the NR gene. As transcription of GS1 and /3-ATPase
were also increased, BAP obviously acted at a transcriptional level and reactivated cellular activity. However,
cytokinins can control genetic expression at posttranscriptional states (Deikman and Hammer, 1995) and
enhanced stability of the mRNA pool may partly contribute to increased mRNA accumulation. In chicory roots,
modification of the NR-mRNA stability could wellfitin
with the results of Northern analysis, which showed a
poor increase of the NR-mRNA level during the first 2 d
of culture with BAP. Cytokinins were shown to enhance
NRA NR-protein and NR-mRNA in materials in which
nitrate reduction was induced by light and nitrate. In
1084
Vuylsteker et al.
tobacco cell suspension cultures or in barley etiolated
leaves they increased NRA which was induced by light
(Lu et al, 1992; Suty et al., 1993); but in both these
cases, the underlying mechanisms differed: in tobacco
cells, kinetin modulated mRNA polyadenylation and its
effects were detectable after 3 days whereas in barley
leaves, BAP enhanced transcription of the NR gene within
min (Lu et al, 1992; Suty et al, 1993). In chicory
detopped roots, which were grown with nitrate over the
course of the experiment, increased transcription of the
NR gene was detectable only 2 d after BAP was added.
The stimulatory effect of cytokinins at the transcriptional
level of NR, may thus be delayed in plant material not
induced by nitrate and NR may be a marker for cell
reactivation by BAP. Such lag phases between the application of cytokinins and the increase of NR transcription
could be explained by considering the BAP effect on
NRA as a consequence of resumed growth of the roots.
In the presence of 10~6 M NAA, in vivo NRA also
increased in excised roots, but to a lesser extent than in
the presence of BAP (seven times on day 5). Moreover,
a 4-fold increase of the NR-protein level occurred at the
4th day while in BAP-treated roots, the level of
NR-protein increased from the 2nd day onwards. NAA
exerted a lower effect on the NR-mRNA level. Thus,
though an increase of NRA level was clearly shown, an
auxin effect at both transcriptional or translational levels
seemed more questionable than the cytokinin effect. In
vitro NR activities assayed with either EDTA or Mg2 +
ions at day 5 favour an important increment of the level
of dephosphorylated NR in the presence of NAA. As
BAP, NAA stimulated growth of the excised roots which
probably contribute to the increasing NR expression.
NRA in either BAP- or NAA-treated excised roots was
always far lower than the NRA values which may be
expected when the NR-protein levels were taken into
account. NRA was shown to be submitted to phosphorylation controls (Kaiser and Huber, 1994; Kojima et al,
1995; MacKintosh et al, 1995; Bachmann et al, 1995).
Whereas staurosporine, a protein kinase inhibitor, could
restore the correlation between NRA and NR-protein
level just after excision (Vuylsteker et al, 1997), it failed
to do it fully in roots grown in the presence of BAP.
Besides dephosphorylation, modification of the carbon
metabolism including several factors such as the cytosolic
redox potential, the energy status, could affect in vivo
NRA, which might become independent of an occasional
increase of the NR-protein level.
Levels of GS1 and NR-mRNAs evolved similarly; they
decreased after the roots were excised and increased upon
addition of either BAP or NAA. GS1 may thus operate
in the nitrate assimilation pathway in chicory roots and
both NR and GS1 might be coregulated. Relationships
between NR and cytosolic GS1 were shown as well, in
the reallocation of nitrogen in the flag leaf of wheat
during grain filling (Peeters and Van Laere, 1994).
In conclusion, both BAP and NAA reinduced NRA in
detopped roots of chicory, which, unlike barley leaves or
tobacco cells used by Lu et al. (1992) and Suty et al
(1993), were fed with nitrate during the whole course of
the experiment. This reinduction partially resulted from
enhanced transcriptional and translational activities.
However, NR activities measured in roots grown with
both growth factors, were lower than activities which
were expected if the level of NR-protein was considered.
Moreover, increment of in vivo NRA in detopped roots
grown with either BAP or NAA never exceeded the NRA
level of undetopped roots, indicating that nitrate assimilation was not tightly correlated to the NR level, but was
also under metabolic control. In this respect, controls
exerted by BAP and NAA probably differ: besides
enhanced transcription and translation, NAA may modify
the phosphorylated status of NR, whereas BAP probably
modifies it to a lesser extent. Reactivation of NRA by
either BAP or NAA was related to hormonal induced
growth. Work is presently underway in order to analyse
histological modifications induced by the hormonal treatments, and to locate where NR is expressed in the different
experimental conditions.
Acknowledgements
We thank Dr G Conejero (INRA, Montpellier) for the gift
of the anti-NR maize polyclonal antiboby; Dr M Caboche for
the gift of the anti-NR maize monoclonal antibody and
Dr T Moureaux (INRA, Versailles) for her helpful assistance
in ELISA determination of NR levels; Dr M Boutry (University
of Leuven) and Dr B Hirel (INRA, Versailles) for the generous
gift of the N. plumbaginifolia /3-ATPsynthase-cDNA and the
N. tabacum glutamine synthetase GSl-cDNA, respectively.
This work was supported by grants from Conseil Regional
Nord-Pas de Calais. C Vuylsteker was supported by a MENESR
fellowship.
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