Influence of BAP and NAA on the expression of nitrate reductase in excised chicory roots
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Journal of
Journal of Experimental Botany, Vol. 43, No. 310, pp. 1079-1085, May 1997 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
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In young planttets of chicory (Cichorium intybus L. var Plants usually reduce nitrate in their leaves where energy
Witloof) nitrate reduction is mainly localized in roots. and reducing power arise (Beevers and Hageman, 1980).
Following root excision, nitrate reductase activity In chicory, a biennial Asteraceae, nitrate reduction occurs
rapidly decreased. This inhibition was first induced by mainly in roots until they differentiate tubers. Thereafter,
phosphorylation of NR, followed by the decrease of nitrate reductase activity is localized in leaves (Dorchies
NR-protein and NR-mRNA contents (Vuylsteker et al., and Rambour, 1985). Roots of young chicory plantlets
1997). Addition of 1 0 " M BAP re-induced NRA in behave as sinks for carbon and sources for nitrogen.
excised roots after 2 d, in vivo NRA reaching 10-fold Nitrate reduction in roots depends on the import of
the level of controls on day 5. This increase of NRA photosynthates which provide carbon skeletons, energy
was related to enhanced NR-protein and NR-mRNA and reductants (Huppe and Turpin, 1994). In plants
contents. After 4 d, BAP increased 16-fold the which reduce nitrate in their roots, tight correlations
NR-protein and 3.5-fold the NR-mRNA levels, respect- between root and shoot metabolism are important.
ively. In vitro NRA measured in the presence of EDTA Nitrate reductase, a key enzyme in the control of
was 5.6-fold higher in BAP-treated roots than in con- nitrogen assimilation, is the target of several regulatory
trol roots confirming the in vivo measurements. The processes. The availability of nitrogen and carbon particu-
high discrepancy between the NR-protein level and larly affects nitrate reductase which is inducible by nitrate,
NRA shows that limiting factors other than the whereas ammonium and amino acids can inhibit its
NR-protein content affected NRA reactivation. With activity (Solomonson and Barber, 1990; Li et al, 1995;
1 0 " M NAA, in vivo NRA was enhanced seven times Sivansankar and Oaks, 1995). Carbon metabolism also
on day 5 and in vitro NRA was increased only 2.5 times. regulates nitrate reductase and, in leaves, the light effect
Enhancement of the NR-protein was more restricted on transcription of NR-mRNA can be mimicked by
with NAA than with BAP. Besides a poor effect at the sucrose (Cheng et al., 1992; Vincentz et al., 1993).
transcriptional level, NAA may control NRA by a phos- Moreover, light controls nitrate reductase activity in
phorylation mechanism underscored by higher sensit- leaves by phosphorylation-dephosphorylation reactions
ivity of in vitro NRA to magnesium. Resumption of NRA (Glaab and Kaiser, 1995; Huber et al., 1992; Kojima
in the presence of either BAP or NAA occurred simul- et al., 1995). In roots, light modulates nitrate reductase
taneously to the increase of the dry weight and was
probably indirectly by photoassimilates (Merlo et al.,
probably driven by increasing needs of reduced
1994). These regulatory processes involving both carbon
nitrogen to support regrowth.
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
Key words: Chicory, BAP, NAA, roots, nitrate reductase. messengers of the nitrogen status towards the root to
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 19971080 Vuylsteker et al.
shoot allocation of biomass (Beck, 1994). Concentration In vivo nitrate reductase activity
of cytokinins within roots and xylem responds to the The roots were harvested at different times, weighed and
nitrogen availability in terms of either total nitrate avail- assayed for NRA according to the in vivo method of Jaworski
(1971). Individual roots were introduced in 2 ml of the
ability (Samuelson and Larsson, 1993) or of differential incubation mixture comprising 62.5 mM KN0 3 , 37.5 mM
distribution of nitrate along the lateral roots (Samuelson K-phosphate buffer pH 7.5 and 1.2% 1-propanol (v/v).
et al., 1995). Their transport is submitted to fluctuations Measurements were made on five independent samples and
of evapotranspiration and hence to foliar activity (Beck, repeated at least three times. The reaction tubes were submitted
1994). They are known to regulate NR activity in response 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
to light or nitrate induction (Lu et al., 1990, 1992; 0.5 ml sulphanilamide (11 mM in 3 M HC1) and 0.5 ml of
Samuelson et al., 1995). In Agrostemma githago embryos, aqueous 10 mM N-\ naphthyl ethylene diamine dichloride.
cytokinins induced NRA only in the presence of ethylene NRA was expressed as nmol nitrite produced min" 1 g" 1 FW.
(Schmerder and Borris, 1986).
Among numerous effects on growth and plant develop- In vitro nitrate reductase activity
ment, auxins stimulate root initiation and the develop- In vitro assays are derived from Merlo et al. (1995). Roots were
ment of lateral roots. Data concerning relationships frozen and ground in a chilled mortar. Extraction buffer
contained 50 mM HEPES-KOH pH 7.5, 5 mM MgCl2, 0.5 mM
between auxins and nitrate reductase are, until now, rare EDTA, 14 mM 2-mercaptoethanol, 0.1% (v/v) Triton X100,
and conflicting (Knypl, 1979). However, a relationship 10% (v/v) glycerol, 50 ^M leupeptin, 0.5 mM PMSF, and 10%
between the development of NRA and the rhizogenic (w/v) polyvinylpyrrolidone. Extracts were then desalted on to a
potential of IAA and IBA on rooting of pea cuttings has G25 Sephadex column equilibrated with the same buffer except
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once been reported (Ahmad, 1988). that it contained neither EDTA nor MgCl2. NRA was assayed
using 50 mM, pH 7.5 HEPES-KOH buffer comprising 10 mM
When roots of young chicory plantlets were excised K.NO3, 0.2 mM NADH, and 10 MM FAD.
and tranferred to liquid medium, nitrate reductase activity Modulation of the activation status of NR in vitro, was
rapidly decreased via a phosphorylation mechanism. performed by adding either 2 mM EDTA or 5 mM MgCl2 to
Thereafter both the NR-protein and NR-mRNA levels desalted extracts. Incubation was performed at 30 °C for 5 min,
decreased. This inhibition was considered as an adaptative and the reaction was then stopped by adding 50 /xl 0.5 M zinc
acetate. Excess NADH was oxidized with phenazine methos-
reduction of the nitrogen assimilatory pathway to a ulphate (final concentration 10fiM). Nitrite was revealed as
senescence-like process, as a result of the suppression of above and NRA activity was expressed as nmol of
the shoot to root correlations (Vuylsteker et al., 1997). nitrite min" 1 mg" 1 protein. The protein content was measured
In sucrose-starved roots, nitrogen assimilation decreased according to Bradford (1976) with bovine serum albumin as a
at the expense of reallocation of reduced nitrogen standard.
(Brouquisse et al., 1991).
ELISA immunoquantification of NR proteins
As low concentrations of cytokinins and auxins have
The NR level was quantified by the two sites ELISA procedure
long been considered to delay senescence (Goldthwaite, according to Cherel et al. (1986) using monoclonal anti NR
1987), the possible effects of both growth factors on the maize 96925 and S6 polyclonal anti NR maize antibodies. These
level of nitrate reductase in excised roots were investi- antibodies were first tested against chicory root NR by Western
gated. Moreover, both hormones are implicated in the blot analysis and immunoprecipitation assays.
control of rhizogenesis and are stored or synthesized in
roots (Torrey, 1976), so they could influence NRA in Total RNA extraction
chicory roots. NRA, NR and NR-mRNA levels were Total RNA was extracted from the root tissues according to a
procedure derived from Chirgwin et al. (1979). One gram tissue
measured in BAP- and NAA-treated excised roots with was ground in liquid nitrogen to a fine powder which was
the purpose of studying their potential reactivating effects. 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
Materials and methods with ethanol precipitation (0.75 vol. ethanol and 0.08 vol. 1 M
acetic acid). Nucleic acids were pelleted and dissolved in 10 mM
Plant material TRIS-HC1 pH 7.5. RNAs were selectively precipitated with 2 M
lithium chloride. RNA were finally dissolved in diethyl pyrocar-
Chicory seeds {Cichorium intybus L. var. Witloof, cv. Flash) bonate treated sterile water.
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 Northern analysis
with a photoperiod of 16/8 h (light/dark) and a light irradiance 20 ng total RNA were run in a 1.5% (w/v) agarose formaldehyde
of 1 4 f i M m " 2 s " ' . After 18 d, plants which developed two gel (Sambrook et al., 1989). Subsequently, blotting was achieved
cotyledons and four leaves were decapitated, and 12 uniform on Hybond-N + (Amersham) membranes. DNA probes were
roots were transferred, in aseptic conditions, into flasks labelled with [a- 32 P]dCTP (111 T Bq mM " ' ICN) using random
containing 50ml HI5 liquid medium. NAA and BAP at a priming (T 7 Quickprime Pharmacia). Hybridizations were
final concentration of 10" 6 M, were aseptically added after performed according to Church and Gilbert (1984); membranes
autoclaving the medium, using 0.20 ^un Acrodiscs®. were then exposed to X-ray films (Kodak X-Omat AR) atHormonal control of nitrate reductase activity 1081
-80°C using intensifying screens. Intensity of the bands was diately after excision and which induced a rapid inhibition
estimated after scanning and digitalization using a Microtek of NRA (Vuylsteker et ai, 1997). After 6d without
Color/Gray scanner (Biorad) connected to a Macintosh LCIII
(Apple) computer. Trie software used was the free ware renewing the medium, roots got brown and NRA finally
NIH-1.56. dropped, reaching the level of NRA in detopped control
The probes were: NR, a partial cDNA from nitrate reductase roots. Nutritional starvation linked to BAP physiological
of Cichorium intybus (X 84102 EMBL Data Library; Palms effects on roots or to hormonal exhaustion can be inferred.
et a]., 1996); GS1, a complete cDNA of cytosolic glutamine In the presence of 10~6 M NAA, the NRA ratio
synthetase of Nicotiana tabacum (gift of B Hirel, unpublished
data); ATPase, a cDNA of a /3 subunit of Nicotiana plumbagini- between NAA-treated roots and control roots reached a
folia (Boutry and Chua, 1985); TUB, cDNA from a tubulin of value of 7 on day 5 and subsequently declined from the
Daucus carota (Borkid and Sung, 1985). 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.
Results
Moreover, stimulation of NRA by 10~6 M NAA was of
In vivo NRA less importance than the enhancement induced by 10 ~6
M BAP.
NRA in roots which were excised from 18-d-old plantlets,
and transferred into liquid medium rapidly decreased Growth
(Vuylsteker et ai, 1997). Addition of either BAP or NAA
in the liquid medium enhanced significantly in vivo NRA Dry weight (DW) of control roots remained stable during
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in detopped roots, 48 h after their transfer (Fig. 1). This the course of the culture (Fig. 2). The loss of nitrate
effect, estimated by the ratio between in vivo NRA in reduction in excised roots was thus partly correlated to
roots treated with a growth factor and NRA in excised the absence of growth and subsequent reduced needs of
control roots, was dependent on the concentration of amino acids. Addition of BAP or NAA to the liquid
both the auxin and the cytokinin. The higher ratios were medium increased DW of roots from, respectively, the
obtained by adding 10~6 M BAP or 10"6 M NAA (data 2nd and the 3rd day on (Fig. 2). After 5 d, DW of BAP-
not shown). or NAA-treated roots were 4-fold higher and 2-fold
In the presence of 10 ~6 M BAP, the values of the NRA higher than the DW of control roots, respectively. The
ratios reached 3 at the 2nd and 10 at the 5th day of the increase of DW occurred simultaneously with enhanced
culture (Fig. 1). On day 5, NRA recovered the level of NRA (Fig. 1).
NRA in intact roots. Thus BAP exerted a long-term
In vitro NRA
effect, resulting in the recovery of NRA measured in
undetopped roots. However, BAP was unable to prevent NRA was shown to be controlled by reversible phos-
the early phosphorylation reaction which occurred imme- phorylation, 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 Fig. 2. Time-course of dry weight of chicory roots in control conditions
at different times after 10~6 M of either BAP or NAA were added. and in the presence of 10~6M of either BAP or NAA. Dry weight was
Enzymatic activities are expressed as: NRA in hormonal conditions/ measured on samples consisting of 12 roots. Means of three repeats are
control NRA. Means±SD (n=\0). shown ± S D .1082 Vuylsteker et al.
to an inhibitor protein. According to these data, models NR-protein
for reversible control of NRA involving interaction of
The level of total soluble proteins per gram fresh weight
phosphorylated NR and nitrate reductase inhibitor
was not significantly affected by NAA and was enhanced
proteins were hypothesized (Kaiser and Huber, 1994;
by only 20% in the presence of BAP, after 5 d (data
MacKintosh et al., 1995; Bachmann et al., 1995).
not shown).
Addition of EDTA to the reaction mixture inhibits the
The level of NR-protein in roots grown with 10~6 M
binding reaction and, consequently, the bulk of NR
BAP, rapidly exceeded the level of NR-protein in roots
protein, phosphorylated or not, contributes to NRA.
of undetopped plantlets. It increased linearly reaching a
Conversely, magnesium stabilizes the inactive phospho-
maximum on day 4, when it was 1 ? times higher than in
NR-NIP complex (MacKintosh et al., 1995). Thus, the
controls; on day 4, it remained 13 times as high as in
ratio between NRA measured in the presence of Mg2 +
controls and subsequently decreased (Fig. 3).
ions and NRA measured in the presence of EDTA
In roots grown with 10~6 M NAA the NR-protein
accounts for the activation rate of NR and reflects the
level increased only from the 3rd day on. Compared to
ratio of active NR-protein.
control excised roots, the NR-protein level was enhanced
When in vitro NRA was first measured in the presence
4 times on day 4, 7.5 times on day 5 and 13 times on day
of either EDTA or Mg 2+ ions, in roots harvested on
6 (Fig. 3).
18-d-old plantlets, the activation rate reached 40%. In
Comparing in vitro NR activities measured with EDTA
excised roots grown for 3 h in the control medium, the
and the levels of NR-protein showed discrepancies
activation rate reached less than 15%, indicating that
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between NR activities and the NR-protein levels. The
excision induced an enhancement of inactive NR. As the
increment of the NR-protein levels induced by both the
assays were carried out without adding any protein phos-
growth factors not only preceded increased NRA, but
phatase inhibitors, the level of inactive NR may be
equally exceeded the increment of NRA. For instance in
underrated. However, as the procedure was carried out
roots grown with BAP, in vitro NRA measured with
rapidly, this may be insignificant. When the growth time
EDTA was 5.6 as high as in controls on day 5, whereas
lasted for 5 d, the activation rate was not measurable,
the NR-protein level was 13 times higher. Similar discrep-
since NRA in the presence of Mg 2+ ions was not detect-
ancies between the NR-protein level and NRA occurred
able. In excised roots of chicory grown for 5 d in the
in roots grown with NAA; whereas the NR-protein level
presence of 10" 6 M of either BAP or NAA, in vitro NR
was 7 times higher than in controls, in vitro NRA was
activities measured in the presence of either EDTA or
only 2.5 times higher on day 5. Thus in both cases, the
Mg 2+ ions were stimulated. On day 5, NRA assayed with
NR-protein level did not match NR activities measured
EDTA was 5.6 times higher in BAP-treated roots than in
in vitro, indicating that part of NR was inactive.
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 400-
Control
reached 34% and 49% in roots grown with BAP and 10"*M NAA
NAA, respectively (Table 1). 10"«M BAP
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. 200-
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 deter-
minations) and were typical of data obtained in three different
experiments.
Before After excision
I 100-
excision
3h 5d
Control Control BAP NAA
Time (hours)
In vitro NRA
with EDTA 12±1.6 10.8±1.2 3.4± 1.6 19±1.3 8.7±0.7 Fig. 3. Time-course of the NR-protein level in excised roots grown in
In vitro NRA the presence of 10~6 M of either BAP or NAA. NR-protein levels in
with MgCl 2 4.9 ±1.4 nd 6.5±1.6 4.3± 1.2 excised roots were quantified by ELISA. NAA and BAP were added
Activation rate (%) 40 nd 34 49 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 1083
Northern blot analysis between days 3 and 4, the /3-ATPase and GS1 mRNAs
levels decreased in NAA treated roots as in control roots.
The mRNA levels of nitrate reductase (NR) cytosolic
However, on day 4, the levels of GS1 and /3-ATPase-
glutamine synthase (GS1) and /3-ATPase decreased in mRNAs remained 2-fold higher in NAA-treated roots
excised control roots indicating that overall transcrip- than in roots grown without NAA.
tional activity probably decreased in the absence of any
growth factor (Vuylsteker et al., 1997).
Conversely, in roots grown with 10~6 M BAP, high Discussion
levels of NR, GS1 and /3-ATPase mRNAs were main- Excision of the roots of young plantlets of chicory and
tained through the time-course of the experiment (Fig. 4). their transfer in a stirred liquid medium, induced a rapid
This was confirmed by scanning the hybridization bands loss of NRA, which was due to phosphorylation of NR.
(Table 2). However, the level of these mRNAs declined This resembled senescence (Vuylsteker et al., 1997).
in control roots grown without BAP, and as a result BAP Indeed, no significant growth was detected in excised
reinduced transcription of the NR, GS1 and /3-ATPase roots. Similarly, in maize, nitrate reduction decreased in
genes. Four days after the transfer in liquid medium senescing detached leaves, in sucrose-starved roots or
containing BAP, compared to control roots of day 4, the after decreasing light intensity delivered to maize plantlets
levels of NR, GSland /9-ATPase mRNAs were increased (Brouquisse et al., 1991; Merlo et al., 1994; Saglio and
3.5, 8.5 and 11.5 times, respectively. The increment of Pradet, 1980).
NR-mRNA occurred simultaneously with the increment Supplying excised chicory roots with either 10~6 M
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of the NR-protein level (Fig. 3). BAP or 10"6 M NAA restored in vivo NRA, 2 d after
Similarly, when excised roots were transferred into they were transferred. Resumption of NRA was related
NAA-containing media, no significant decrease of the to the induction of growth in roots grown with either
NR-mRNA levels was observed (Table 2). Conversely, BAP or NAA. The inference was that the NRA increase
was driven by needs of nitrogen essential for growth
Day 3 Day 4 resumption. In vitro NRA increased as well, but to a
lesser extent. A similar discrepancy between in vivo and
C B N C B N in vitro NRA has already been reported (Oaks, 1992).
Moreover, in vitro NR activities did not match the
NR
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,
ATP synthase Arabidopsis thaliana mutants impaired in the expression
of NR and fed with nitrate as the sole source of nitrogen
TUB 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
GS1
increased and declined concurrent with the level of
NR-mRNA, both reaching a peak on day 4. NRA
Fig. 4. Northern blot analysis of total RNA of excised chicory roots remained unchanged during the course of the first day of
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. the culture, and subsequently increased reaching a max-
C, control; N, NAA; B, BAP. Probes were cDNAs of nitrate reductase imum on day 5. On day 4, the levels of NR-mRNA and
(NR), /3-subunit of ATP synthase (ATP synthase), a-tubulin (TUB), NR-protein were 3.5 and 15 times as high as in controls,
cytosolic glutamine synthetase (GS1).
respectively. Thus BAP induced enhanced transcription
of the NR gene. As transcription of GS1 and /3-ATPase
Table 2. Densitometric quantification of mRNAs evidenced in were also increased, BAP obviously acted at a transcrip-
Fig. 4 tional level and reactivated cellular activity. However,
Data are given in arbitrary units. Intensity of the different bands of the cytokinins can control genetic expression at post-
controls on day 3 were arbitrary set at 100. As the signal with the transcriptional states (Deikman and Hammer, 1995) and
tubulin probe was very weak, an accurate determination of the tubulin
expression was prevented. enhanced stability of the mRNA pool may partly contrib-
ute to increased mRNA accumulation. In chicory roots,
3d 4d modification of the NR-mRNA stability could wellfitin
Probes Control BAP NAA Control BAP NAA
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
888
NR 140 120 40 140 90
ATP synthase 260 210 20 230 40 NRA NR-protein and NR-mRNA in materials in which
GS1 180 220 20 170 50
nitrate reduction was induced by light and nitrate. In1084 Vuylsteker et al.
tobacco cell suspension cultures or in barley etiolated In conclusion, both BAP and NAA reinduced NRA in
leaves they increased NRA which was induced by light detopped roots of chicory, which, unlike barley leaves or
(Lu et al, 1992; Suty et al., 1993); but in both these tobacco cells used by Lu et al. (1992) and Suty et al
cases, the underlying mechanisms differed: in tobacco (1993), were fed with nitrate during the whole course of
cells, kinetin modulated mRNA polyadenylation and its the experiment. This reinduction partially resulted from
effects were detectable after 3 days whereas in barley enhanced transcriptional and translational activities.
leaves, BAP enhanced transcription of the NR gene within However, NR activities measured in roots grown with
min (Lu et al, 1992; Suty et al, 1993). In chicory both growth factors, were lower than activities which
detopped roots, which were grown with nitrate over the were expected if the level of NR-protein was considered.
course of the experiment, increased transcription of the Moreover, increment of in vivo NRA in detopped roots
NR gene was detectable only 2 d after BAP was added. grown with either BAP or NAA never exceeded the NRA
The stimulatory effect of cytokinins at the transcriptional level of undetopped roots, indicating that nitrate assimila-
level of NR, may thus be delayed in plant material not tion was not tightly correlated to the NR level, but was
induced by nitrate and NR may be a marker for cell also under metabolic control. In this respect, controls
reactivation by BAP. Such lag phases between the applica- exerted by BAP and NAA probably differ: besides
tion of cytokinins and the increase of NR transcription enhanced transcription and translation, NAA may modify
could be explained by considering the BAP effect on the phosphorylated status of NR, whereas BAP probably
NRA as a consequence of resumed growth of the roots. modifies it to a lesser extent. Reactivation of NRA by
In the presence of 10~6 M NAA, in vivo NRA also either BAP or NAA was related to hormonal induced
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increased in excised roots, but to a lesser extent than in growth. Work is presently underway in order to analyse
the presence of BAP (seven times on day 5). Moreover, histological modifications induced by the hormonal treat-
a 4-fold increase of the NR-protein level occurred at the ments, and to locate where NR is expressed in the different
4th day while in BAP-treated roots, the level of experimental conditions.
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 Acknowledgements
auxin effect at both transcriptional or translational levels We thank Dr G Conejero (INRA, Montpellier) for the gift
seemed more questionable than the cytokinin effect. In of the anti-NR maize polyclonal antiboby; Dr M Caboche for
vitro NR activities assayed with either EDTA or Mg2 + the gift of the anti-NR maize monoclonal antibody and
ions at day 5 favour an important increment of the level Dr T Moureaux (INRA, Versailles) for her helpful assistance
of dephosphorylated NR in the presence of NAA. As in ELISA determination of NR levels; Dr M Boutry (University
of Leuven) and Dr B Hirel (INRA, Versailles) for the generous
BAP, NAA stimulated growth of the excised roots which gift of the N. plumbaginifolia /3-ATPsynthase-cDNA and the
probably contribute to the increasing NR expression. N. tabacum glutamine synthetase GSl-cDNA, respectively.
NRA in either BAP- or NAA-treated excised roots was This work was supported by grants from Conseil Regional
always far lower than the NRA values which may be Nord-Pas de Calais. C Vuylsteker was supported by a MENESR
expected when the NR-protein levels were taken into fellowship.
account. NRA was shown to be submitted to phosphoryl-
ation controls (Kaiser and Huber, 1994; Kojima et al, References
1995; MacKintosh et al, 1995; Bachmann et al, 1995).
Whereas staurosporine, a protein kinase inhibitor, could Ahmad A. 1988. Nitrate accumulation and nitrate reductase
restore the correlation between NRA and NR-protein activity during rooting of pea cuttings treated with auxins.
Indian Journal of Experimental Biology 26, 470—2.
level just after excision (Vuylsteker et al, 1997), it failed Bachmann M, McMichael Jr RW, Huber JL, Kaiser WM,
to do it fully in roots grown in the presence of BAP. Huber SC. 1995. Partial purification and characterization of
Besides dephosphorylation, modification of the carbon a calcium-dependent protein kinase and an inhibitor protein
metabolism including several factors such as the cytosolic required for inactivation of spinach leaf nitrate reductase.
redox potential, the energy status, could affect in vivo Plant Physiology 108, 1083-91.
NRA, which might become independent of an occasional Beck E. 1994. The morphogenic response of plants to soil
nitrogen: adaptative regulation of biomass distribution and
increase of the NR-protein level. nitrogen metabolism by phytohormones. In: Detlef-Schulze E,
Levels of GS1 and NR-mRNAs evolved similarly; they ed. Flux control in biological systems from enzyme to population
decreased after the roots were excised and increased upon and ecosystems. New York: Academic Press, 119-51.
addition of either BAP or NAA. GS1 may thus operate Beevers L, Hageman RH. 1980. Nitrate and nitrite reduction.
in the nitrate assimilation pathway in chicory roots and In: Stumpf PK, Conn EE, eds. The biochemistry of plants.
New York: Academic Press, 115-68.
both NR and GS1 might be coregulated. Relationships Borkid C, Sung ZR. 1985. Expression of tubulin genes during
between NR and cytosolic GS1 were shown as well, in somatic embryogenesis. In: Terzy M, Pitto L, Sung ZR,
the reallocation of nitrogen in the flag leaf of wheat eds. Workshop held at San Antonio, Rome: Incremento
during grain filling (Peeters and Van Laere, 1994). Produttivita Risorse Agricole, 14-21.Hormonal control of nitrate reductase activity 1085
Boutry M, Chua NH. 1985. A nuclear gene encoding the beta Lu J-l, Ertl JR, Chen C-m. 1990. Cytokinin enhancement of
subunit of the mitochondrial ATP synthase in Nicotiana the light induction of nitrate reductase transcript levels in
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