Journal of Experimental Botany, Vol. 51, No. 344, pp. 539-546,
March 2000
© 2000 Oxford University Press
Nitrate assimilation in chicory roots (Cichorium intybus L.) which acquire radial growth
1 Laboratoire de Physiologie et Génétique Moléculaire Végétales, Université des Sciences et Technologies de Lille, Bâtiment SN2, F-59655 Villeneuve d'Ascq Cedex, France
2 Laboratoire du Métabolisme et de la Nutrition des Plantes, INRA, Route de St Cyr, F-78026 Versailles, France
Received 23 July 1999; Accepted 14 October 1999
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Abstract |
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Nitrate assimilation was analysed in chicory plants (Cichorium intybus L. cv. Turbo) during the early vegetative growth. Nitrate reductase (NR, EC 1.6.6.1) activity (NRA) was measured in roots and leaves at different developmental stages. During phase I, which corresponds to the structural growth (21–42 DAS), nitrate reduction mainly occurred in the roots. At the onset of the tuber formation (phase II), which is characterized by the formation of a cambium inducing a radial growth (42–63 DAS), NRA rapidly decreased in roots and developed in leaves. A tight correlation was found between the nitrate content, the amino acid level and NRA in roots and leaves. Northern blot and ELISA analysis showed that both levels of NR mRNA and NR protein were not modified during the time-course of the experiment suggesting that modification of nitrate assimilation was not controlled at a transcriptional level. In vitro NRA assayed in presence of either Mg2+ ions or EDTA showed that NR was influenced at least in part by a reversible phosphorylation/dephosphorylation reaction. Okadaic acid, a serine–threonine protein phosphatases inhibitor, strongly decreased NRA. Conversely, staurosporine, a serine–threonine protein kinases inhibitor, did not significantly change NRA in roots or leaves. Therefore, NRA was regulated at a post-translational level during the early vegetative growth by modifying the phosphorylation balance of the NR protein in chicory.
Key words: Chicory, nitrate reductase, phosphorylation, regulation, tuber formation.
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Introduction |
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Plants assimilate nitrogen as a source for growth, biomass production and development. Nitrogen is mainly absorbed as nitrate which is the most common nitrogen source available to higher plants. Nitrate assimilation is the primary pathway by which reduced nitrogen is accumulated in plants. That involves the consecutive action of two enzymes: nitrate reductase (NR), a cytosolic enzyme which reduces nitrate to nitrite using NAD(P)H as electron donor, and nitrite reductase (NiR), a plastidic enzyme which reduces nitrite to ammonium. Nitrate reduction and its further assimilation represent a major metabolic function (Crawford et al., 1992
). NRA is hence considered as a limiting factor for higher plant protein production, development and growth (Huppe and Turpin, 1994). There is a general agreement that distribution of nitrate assimilation between roots and shoots is species-dependent. Nitrate reduction is mainly performed in roots of woody plants and temperate origin species as compared to herbaceous plants (Andrews et al., 1984; Gojon et al., 1994). The partitioning of nitrate assimilation between roots and shoots is also dependent on exogenous nitrate availability. However, in most higher plants, nitrate is reduced more efficiently in leaves than in roots because of the readily available reductants, energy and carbon skeletons produced by photosynthesis (Beevers and Hageman, 1980; Solomonson and Barber, 1990; Oaks, 1994). NRA and other enzymatic activities which are involved in the nitrate assimilation pathway also change during organ and plant development (Andrews, 1986; Kenis et al., 1992). Distribution of carbohydrates and nitrogenous compounds between aerial and subterranean parts of a plant is thus of central importance for plant growth and development. It depends upon the capability of cells, tissues or organs to overproduce, export or import and metabolize reduced carbon and nitrogen.
Chicory (Cichorium intybus L. var. Witloof cv. Turbo), a biennial species belonging to the Asteraceae, provides an interesting model for studying spatial and temporal regulation of nitrate assimilation (Palms et al., 1996). During the first year of its culture, a first phase extending over 1.5–2 months after sowing consists of structural growth producing a plant comprising a long primary root with many laterals and a rosette of 6–8 leaves. During a second phase extending from the 2nd to the 5th month, radial growth of the tap root becomes prevalent producing a mature tuberous root accumulating inulin and nitrogen compounds (vegetative storage proteins, amino acids) which constitute up to 80% and 1% of the dry weight, respectively (Limami et al., 1993, 1996). Finally, from the 5th month on, a senescence phase begins accompanied by the cessation of fructan and N-compounds accumulation in the tuber (Ameziane et al., 1997a). Work on chicory has been done on seasonal partitioning of compounds between tuberous roots and shoots that results in fluctuations in the soluble nitrate, amino acid, protein, and carbohydrate pools within the roots accompanied by modifications in the enzymatic activities involved in the nitrate assimilation pathway (Sechley et al., 1991).
The aim of the present work was to investigate the partitioning of nitrate assimilation in younger chicory plants, at the transition from phase I to phase II. NRA, nitrate content, the level of NR-mRNA, and the level of NR protein were measured in roots and shoots during phase I and the early phase II in order to investigate the biochemical and molecular processes underlying this transition. Moreover, NRA in the presence of protein phosphatases and protein kinases inhibitors were performed in order to analyse whether a post-translational regulation occurred.
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Materials and methods |
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Plant material
Chicory seeds (Cichorium intybus L. var. Witloof cv. Turbo) were germinated in Petri dishes on distilled water in growth chambers maintained at 20±1 °C with a photoperiod of 12/12 h (light/dark) and a light irradiance of 50 µM m-2 s-1. Once germinated, the seedlings were transferred in tanks containing Perlite, sprinkled with a solution of Heller's salts containing 7 mM KNO3 (Heller, 1953
) and placed in the growth chambers under experimental conditions as described above. Plants at 21, 35, 42, 49, 56, and 63 DAS were sampled. Shoots and roots were excised separately for analysis.
Nitrate reductase assays
The in vivo NR activity was measured according to Jaworski (Jaworski, 1971). Root and leaf samples (200 mg) were placed in 2 ml of the incubation mixture containing 0.1 M K-phosphate buffer (pH 7.5), 0.1 M KNO3 and 1.2% 1-propanol for 30 min at 28 °C in the dark. The colorimetric determination of the reaction was achieved by adding 1 ml of sulphanilamide (11 mM in 3 N HCl) and 1 ml of aqueous 10 mM N-1-naphthyl-ethylene-diamine-dihydrochloride. Activities are expressed as nmol 
released g-1 FW min-1.
In vitro assays are derived from Merlo et al. (Merlo et al., 1995). One gram of frozen root or leaf tissues were ground to a fine powder with liquid nitrogen in a chilled mortar, and suspended in 2 ml of 50 mM HEPES-KOH buffer, pH 7.5 containing 5 mM MgCl2, 0.5 mM EDTA, 14 mM ß-mercaptoethanol, 0.1% (v/v) Triton X-100, 10% glycerol, 50 mM leupeptin, 0.5 mM PMSF, and 10% (w/v) Polyclar AT. The homogenate was centrifuged for 20 min at 5000 g and 0.1 ml of supernatant was supplemented with 0.4 ml of 50 mM HEPES-KOH buffer pH 7.5 which contained 10 mM KNO3, 0.2 mM NADH, 10 µM FAD. Modulation of the activation status of NR was performed by adding either 2 mM EDTA or 5 mM MgCl2. Incubation was performed for 5 min at 30 °C and the reaction was stopped by adding 0.05 ml of 0.5 M Zn acetate. Excess NADH was oxidized with phenazine methosulphate (final concentration 10 mM). Nitrite was revealed as above and NR activity was expressed as nmol released min-1 mg-1 protein. The protein content was measured using bovine serum albumin as a standard (Bradford, 1976).
Inhibitors
Staurosporine was dissolved in DMSO at a concentration of 50 µg ml-1, stored at -20 °C, and used at a final concentration of 0.1 nM. Okadaic acid was dissolved in ethanol at a concentration of 25 µg ml-1, and used at a final concentration of 0.5 µM. All chemicals were from Sigma Chemical Co., St Louis, MO.
ELISA immunoquantification of NR proteins
The NR level was quantified by the two sites ELISA procedure according to Chérel et al. (Chérel et al., 1986) using a 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.
Nitrate determination
Tissues (100 mg) were lyophilized and were incubated for 1 h at 45 °C in 1 ml of bi-distilled water. The suspension was centrifuged for 15 min at 5000 g and the nitrate level was measured in the supernatant according to Cataldo et al. (Cataldo et al., 1975). Absorbance was measured at 410 nm.
Extraction of RNA and Northern blot analysis
Total RNA was extracted from leaf or root tissues frozen in liquid nitrogen according to a procedure derived from Verwoerd et al. (Verwoerd et al., 1989). Denatured RNA samples were electrophoresed on 1.5% agarose formaldehyde gel (Sambrook et al., 1989) and blotted on Bioprobe-Hybond-N+ membrane. DNA probe was labelled with 
-32P-dCTP (111 Tbq mM-1-ICN) using random priming (T7 Quick Prime Pharmacia). Hybridization was performed at 65 °C according to Church and Gilbert (Church and Gilbert, 1984) with a NR-cDNA (cNRS) chicory as a probe. Membrane was then exposed to Kodak X-Omat AR film for 48 h.
Amino acid analysis
Amino acids were successively extracted from 100 mg of dry tissue with 80% then 50% ethanol and finally with water at 5 °C. Then they were separated on a Biotronik LC5001 analyser by ion exchange chromatography, identified using a standard mixture of amino acids (Benson standard PANB), and finally quantified using the PE Nelson 2100 software (Rochat and Boutin, 1989).
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Results |
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Nitrate reductase activity during the photoperiod
In order to set up a specific time point of harvesting, the fluctuations of NRA during the day were analysed. In vivo NRA was measured in 3-week-old chicory plants over the course of the 12/12 h light/dark cycle. NRA showed important diurnal changes in leaves and roots tissues (Fig. 1
). In vivo NRA gradually increased in roots and leaves within the 3 h following the light transition and declined thereafter. Therefore, in subsequent experiments, NRA was assayed 3 h after the light period was set on.
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) of chicory plants during the photoperiod. Data are the mean values of three replicates
Changes of leaf and root biomass during early vegetative growth
Production of biomass in the roots and leaves of plants grown with 7 mM 
was analysed during plant development (Fig. 2a, b). In both organs, the biomass expressed as dry weight increased slowly until 49 DAS and more significantly thereafter. Sixty-three DAS, dry weight of chicory roots and leaves were, respectively, about 7- and 10-fold higher than in 49-week-old plants. The leaf : root DW ratio was initially low, increased thereafter, and remained stable until 49 DAS (Fig. 2c). The highest amount of dry matter accumulated in leaves at 63 DAS and consequently increased the leaf/root DW ratio at that time.
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Nitrate assimilation during chicory plant development
In vitro NRA was measured in roots and leaves of chicory at different harvesting periods in the presence of either Mg2+ ions or EDTA. Sensitivity of in vitro NRA towards magnesium is currently referred as an estimation of the level of inactivated NR. According to data from MacKintosh et al. (MacKintosh et al., 1995), Mg2+ ions promote the linkage between active phophorylated NR and NIP (nitrate reductase inhibitor protein), an inhibitor belonging to the 14-3-3 protein family (Moorhead et al., 1996). EDTA chelates divalent cations and thus prevents the formation of the inactive complex. Thus, measurement with EDTA reflects potential NRA depending on the level of active NR under either a dephosphorylated or phosphorylated form. Consequently, the ratio between NRA measured with Mg2+ ions and NRA measured with EDTA, refects the ratio between active NR and total NR.
In both enzymatic assays, NRA in chicory roots increased during phase I reaching maximal activity 42 DAS. Then, activities declined gradually during phase II (Fig. 3a, b). In leaves, NRA in both assays continuously increased as the plants developed. Forty-nine DAS, NRA prevailed in leaves over NRA in the roots (Fig. 3a, b). In young plantlets (21 DAS), NRA measured in the presence of Mg2+ ions was probably severely inactivated since measurements could not be carried out.
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Measured by ELISA analysis, the level of NR protein, which was higher in roots than in leaves, remained stable during the development of the chicory plants (Table 1). Modifications of NRA during the development was not related to changes in either the level of NR protein or to its degradation.
Effect of okadaic acid and staurosporine
It was decided to verify the hypothesis of a phosphorylation effect by using okadaic acid which is a specific inhibitor of protein phosphatases of the 1 and 2A types of vertebrates, yeasts and plants (Cohen et al., 1990) and staurosporine which inhibits various serine and threonine protein kinases by competing with ATP (MacKintosh and MacKintosh, 1994). Addition of 0.5 µM okadaic acid to the incubation buffer, strongly decreased NR activation state obtained by comparison of NRA assayed in the presence of EDTA and Mg2+. As NRA was severely lowered in the presence of okadaic acid, some measurements of the NR activation state could not be carried out in the presence of Mg2+ (Table 2). In the presence of staurosporine, NRA was not significantly modified when assayed with EDTA or Mg2+ and the NR activation state was close to the control (Table 2).
Nitrate partitioning during plant development
In order to find out a possible correlation between the nitrate level and NRA, determination of the level of nitrate was carried out in roots and leaves collected during plant development (Fig. 4).
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The nitrate level roughly fitted the NRA patterns in either roots or leaves. The nitrate content of the chicory roots increased until 42 DAS (3.5 mg
DW), and subsequently decreased during phase II reaching a minimal value of 63 DAS. The foliar nitrate levels increased regularly during the plant development with a peak observed in 63-d-old plants (6.2 mg DW versus 3 mg DW in roots).
Amino acid analysis
It is well established that final products of nitrogen assimilation such as glutamine and asparagine could inhibit the expression of NR (Li et al., 1995; Sivasankar and Oaks, 1996). In order to verify whether the level of some amino acid modified NRA, analysis of the amino acid levels in roots and leaves was performed during the plant growth (Table 3). The amino acid level was 2-fold higher in roots of 56 DAS (phase II) compared to 21 DAS (phase I). Glutamate, glutamine and aspartate represented approximately 65–70% of the total amino acid content. The level of arginine, which is stored in mature tuberous roots (Limami et al., 1993), was very low during the time-course of the experiment. In leaves, the total amino acid content remained higher than in roots (Table 1). The relative abundance of most of the amino acids did not significantly differ during the time-course of the experiment except glutamine and asparagine. Moreover, the level of serine in leaves was higher than in roots.
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-butyric acid.
Regulation of NR mRNA level
The molecular mechanism underlying NRA regulation in tissues during plant growth was investigated. Northern blot analysis was performed on roots and leaves using a chicory nia probe (Fig. 5). Contrary to NRA, NR mRNA levels did not reveal any modification during the time of the experiment, indicating that, in both organs, the nia gene was continuously expressed. This suggests that fluctuations in NRA did not proceed from modified transcriptional activity of the nia gene.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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A variety of environmental and developmental stimuli regulate nitrate assimilation. In chicory plants exposed to light, NRA increased about 10-fold within the first 3 h. This supports previous observations which suggested that NRA undergoes rapid changes a few hours after the plants were subjected to light transitions (Galangau et al., 1988
; Bowsher et al., 1991).
Besides light, numerous other factors influence the expression of NR. Although most higher plants express NR in photosynthetic tissues, temperate non-legume species vary greatly in their partitioning of nitrate assimilation between root and shoot according to the age of the seedlings (Andrews, 1986). In chicory grown in a growth chamber, NRA which evolved according to the pattern of the nitrate content of the cells, was higher in roots than in leaves until 42 DAS (phase I) confirming previous data in field-grown plants (Dorchies and Rambour, 1985). This suggests that in chicory, as in other slowly growing plants (Gojon et al., 1994), the young roots support the major part of nitrogen assimilation and hence represent a source of nitrogenous compounds for leaves. At the onset of phase II, nitrate reduction shifted towards the leaves. As a result, the primary root of chicory which acted as a source of nitrogen during phase I, progressively developed as a sink during phase II. At this stage of the vegetative growth (21–63 DAS), arginine which was shown to accumulate in mature roots (Ameziane et al., 1997b) was present in only a small amount. Higher levels of serine in leaves than in roots may be ascribed to photorespiration. The level of glutamate, glutamine and asparagine increased up to 56 DAS and thereafter declined in the roots, whereas it always increased in leaves. Glutamine, a product of the nitrate assimilatory pathway, and an intermediate for the synthesis of other amino acids, has been found to act negatively on the expression of NRA. Moreover, addition of glutamine to the nutrient solution strongly reduced the accumulation of NR mRNA in tobacco or maize roots, but had only minor effects in the leaves (Deng et al., 1991; Li et al., 1995). It therefore appears that glutamine may be important in the regulation of the expression of NR in root tissues, but not necessarily in shoot tissues. Although the level of glutamine was raising in the roots, Northern analysis showed no modification of the NR mRNA level. This differed from earlier data which showed an inductive effect of nitrate on chicory plants transferred from a medium without nitrate to a nitrate-containing one (Palms et al., 1996). In the experimental conditions used here nitrate was supplied daily, so that the nia gene might be induced continuously. However, since glutamine was shown to inhibit nitrate absorption (Touraine et al., 1994; Muller et al., 1995) such an effect cannot be ruled out in chicory.
Since both the levels of NR mRNA and NR protein were not modified during the time-course of the experiment, metabolic regulatory processes were involved.
A reversible phosphorylation/dephosphorylation reaction involved in the control of NRA is nowadays unequivocally demonstrated (Kaiser and Huber, 1994; McKintosh et al., 1995; Kaiser et al., 1999).
Based on the sensitivity of phospho-NR to Mg2+ ions inhibition (Huber et al., 1994), NRA assayed in chicory was influenced by such a reversible reaction. The NR activation state decreased in the roots at the onset of phase II whereas it progressively and continuously increased in the leaves during early vegetative growth. NRA measured after extraction with an excess of Mg2+ ions, may reflect the in vivo activity (Kaiser and Huber, 1997). Therefore the evolution of the activation states during the time of the experiment probably reflected the actual enzyme activities in both the roots and leaves. The NR activation state was not modified when NRA was assayed in the presence of staurosporine, except in 21-d-old roots where it was lower than at the subsequent sampling stages. This suggest that, except in the 21-d-old roots, protein kinases did not modify the total NRA probably because the inactive complex phospho-NR-NIP was readily dissociated by the phosphatases.
Conversely the activation state of NR in the presence of okadaic acid was strongly decreased in the roots throughout the time of the experiment. This is in agreement with data obtained previously which showed a strong decrease of NRA in the presence of okadaic acid in detopped chicory roots (Vuylsteker et al., 1997).
Taken all together these data fit in with results which showed that auxiliary proteins such as phosphatases, kinases or the NR inhibitor protein (NIP) which modulate NR are probably expressed independently of NR (Glaab and Kaiser, 1996). However, based on in vitro NRA assayed in the presence of either staurosporine or okadaic acid, it can be assumed that phosphatases are particularly active in controlling NRA in the roots of these young chicory plants.
In conclusion, reorientation of the growth axis and acquisition of storage functions of the chicory root were accompanied by strong physiological modifications. As the cambium was formed and the thickening of the tap root was begun, NRA shifted from the root towards the aerial rosette which was growing up. This shift did not change the NR mRNA level at least during the time of the experiment, indicating that either the nia gene was continuously induced or that post-translational control mechanisms were operating.
In young chicory developing roots, NRA is probably regulated by a phosphorylation–dephosphorylation mechanism, but a negative feedback of glutamine on NRA cannot be ruled out. Moreover, in the mature tuberous root as fructans accumulate (Ameziane et al., 1995), a competition for carbon skeletons and energy between the sucrose metabolism and the nitrate assimilatory pathways may intervene. Indeed, such a competition between both these pathways are now widely accepted (Huppe and Turpin, 1994).
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Acknowledgments |
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The authors thank Viviane Thint for technical assistance.
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Notes |
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3 To whom correspondence should be addressed. Fax: +33 3 20 33 60 44. E-mail:rambour{at}univ\|[hyphen]\|lille 1.fr
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Abbreviations |
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DAS, day after sowing; NR, nitrate reductase; NRA, nitrate reductase activity..
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