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Journal of Experimental Botany, Vol. 48, No. 306, pp. 59-65, January 1997 Journal of Experimental Botany Nitrate reductase activity in chicory roots following excision Christophe Vuylsteker, Brigitte Huss and Serge Rambour1 Laboratoire de Physiologie et Genetique Moteculaire V6g4tales, Universite des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq Cedex, France Received 15 April 1996; Accepted 24 July 1996 Abstract In young chicory plantlets [Cichorium intybus L. Witloof cv. Flash), nitrate assimilation takes place mainly in the roots. Nitrate reductase activity (NRA) was measured in roots deprived of shoot control by excision and transferred into a sucrose-containing medium. Such a treatment resulted in a drop of about 6 0 % of NRA within 3 h. The level of NR protein decreased after 12 h and the level of NR-mRNA after several days. This adaptation of nitrate assimilation to excision was affected by a phosphorylation-dephosphorylation mechanism as shown by increased sensitivity to magnesium of in vitro NRA. Okadaic acid, a serinethreonine protein phosphatases inhibitor, enhanced the decrease of NRA. Conversely, staurosporine, a serine-threonine protein kinases inhibitor, antagonized the inhibition of NRA. This suggests that excision caused a rapid inactivation of NRA in roots of chicory by modifying the phosphorylation balance towards a phosphorylated NR form which could enter an inactive complex. Key words: Chicory, nitrate reductase, staurosporine. Introduction As much as 25% of the energy of photosynthesis is consumed by the nitrate assimilation pathway (Solomonson and Barber, 1990). As a consequence, most of the fast growing plants reduce nitrate in their leaves where the main part of the reducing power arises directly from light via ferredoxin (Beevers and Hageman, 1980). Thus nitrate reduction does not compete with the photoreduction of CO2 (Robinson, 1988). Because of such a high energy requirement, regulation of NR is often viewed as an energy balance between nitrate assimilation and CO2 reduction (Oaks, 1985). This scheme is supported by data from Cheng et al. (1992) and Vincentz et al. (1993) who showed that expression of reporter genes directed by 5' flanking regions of NR genes is regulated by sucrose in the absence of light. Conversely glutamine inhibits the induction of NR in tobacco and Nicotiana plumbaginifolia (Deng et al., 1991; Vincentz et al., 1993). When the nitrate assimilatory pathway occurs in the roots, high amounts of photosynthates must be imported and oxidized to provide the required reductants, energy and carbon skeletons. It has been assumed that the mechanisms involved in the control of nitrate reduction were similar in leaves and roots. Since the heterotrophic status of the roots imposes a total dependence of nitrate reduction on photosynthates supplied by the leaves, the relationship between NRA and light or photosynthesis remains indirect compared to leaves (Huppe and Turpin, 1994). This implies some differences in the control mechanisms. However, fewer studies have been done on the metabolic regulation of nitrate reduction and assimilation in roots, particularly in the carbohydrate storing ones. Chicory which is a biennial Asteraceae, develops a rosette of leaves and a tuberous root at the end of the first year of its growth cycle. It provides an interesting model for studying spatial and temporal regulation of nitrate assimilation. In chicory, in vivo NRA remains higher in roots than in leaves whether the plants are grown in vitro or in fields until they become tuberous (Dorchies and Rambour, 1985). The nitrate content is high in roots during this period and accounts for almost 10% of the total nitrogen (Limami et al., 1993). During tuberization, which starts at approximately the third month, NRA decreases in roots and leaves supply the 1 To whom correspondence should be addressed. Fax: +33 20 43 68 49. E-mail: rambour©univ-Lille1 .fr Abbreviations: ATPase, ATP synthase; GS, glutamine synthetase; NR, nitrate reductase; NRA, nitrate reductase activity; TUB, tubulin. © Oxford University Press 1997 60 Vuylsteker et al. plant with reduced nitrogen (Dorchies and Rambour, 1985). Other enzymatic activities involved in the nitrate assimilation pathway such as glutamate synthase or glutamine synthetase show similar seasonal modifications (Sechley et al., 1991). At that later stage, roots accumulate inulin and reduced nitrogen, predominantly as amino acids whereas their nitrate content becomes insignificant (Limami et al., 1993). Thus, nitrate reduction in chicory is controlled by the nitrate flux and is tightly linked to modifications between sink and source strength during the developmental cycle. In roots, NRA is controlled by NR synthesis in response to nitrate (Oaks, 1985), cytokinins (Hanisch Ten Cate and Breteler, 1982), or by NR proteolysis (Poulle et al., 1987). In maize roots, glutamine exerts a particularly strong inhibition on NR at the transcriptional level (Li et al., 1993). Recent evidence shows that NR is under metabolite control that is exerted by a reversible phosphorylation/dephosphorylation reaction. In leaves, dark decreases NRA by inhibiting dephosphorylation of the NR protein (Kaiser and Huber, 1994) whereas in roots maintained in the dark, the decrease of NRA is related rather to slow protein turnover than to slow dephosphorylation (Glaab and Kaiser, 1993). Conversely, phosphorylation of root NR occurs in response to anaerobiosis (Glaab and Kaiser, 1993; Kaiser et al., 1993). Recent data demonstrated that NR inactivation is achieved by the formation of a complex comprising phosphorylated NR and an inhibitor protein (Kaiser and Huber, 1994; MacKintosh et al., 1995; Bachmann et al., 1995). Dephosphorylation releases active NR and the inhibitor protein. Detopping plantlets abolishes the shoot-to-root relationships. In the absence of sucrose, nitrate reduction is rapidly decreased (Brouquissee/o/., 1992). This resembles senescence in leaves. The decrease of enzymatic activities related to nitrogen assimilation and the subsequent increases of protein and amino acid degrading activities are metabolic adaptations to maintain respiratory activity (Brouquisse et al., 1991; Saglio and Pradet, 1980). Root excision and their transfer into a liquid medium containing sucrose should abolish the shoot control on nitrate reduction without creating a carbohydrate starvation. It could be inferred that excised chicory roots supplied with sucrose constitute a system in which nitrate reduction is only determined by the nitrogen demand of the root. A rapid inactivation of NR by a phosphorylation reaction immediately after the roots were detopped is reported here. Thereafter, the levels of both NR protein and NR-mRNA were strongly decreased. Materials and methods Plant material Chicory seeds {Cichorium intybus L. var. Witloof, cv. Flash) were surface-sterilized and germinated on solid growth medium H15, containing 15 mM sucrose, salts of Heller (1953) and 7 g P ' agar. The growth chamber was maintained at 22±1 °C with a photoperiod of 16/8 h (light/dark) and a light irradiance of 14 ^M m " 2 s " ' . After 18 d, plants which developed two cotyledons and four leaves were decapitated, and twelve uniform roots were transferred, in aseptic conditions, into flasks containing 50 ml H15 liquid medium. Inhibitors Staurosporine was dissolved in DMSO at a concentration of 50/xgml" 1 , stored at — 20 °C, and used at a final concentration of 50ngml"'. Okadaic acid was dissolved in a solution made of ethanol and liquid medium (HI5) (1 : 1, v/v) at a concentration of 25/i.gml" 1 , and used at a final concentration of 0.8/igmr 1 . All chemicals were from Sigma Chemical Co., St Louis, MO. In vivo nitrate reductase activity The roots were harvested at different times, weighed and assayed for NRA according to Jaworski (1971). Individual roots were introduced in 2 ml of the incubation mixture comprising 62.5 mM KNO 3 (5 vols), 37.5 mM K-phosphate buffer pH 7.5 and 1.2% 1-propanol (v/v). Measurements were made on five independent samples. Experiments were repeated at least twice. Nitrite was revealed by adding 0.5 ml (11 mM in 3 M HC1) and 0.5 ml of aqueous 10 mM jV-1 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-K.OH pH 7.5, 5 mM MgCl2) 0.5 mM EDTA, 14 mM 2-mercaptoethanol, 0.1% (v/v) Triton XI00, 10% (v/v) glycerol, 50,xM leupeptin, 0.5 mM PMSF, and polyvinylpyrrolidone 10% (w/v). Extracts were desalted on G25 Sephadex columns equilibrated with the same buffer without EDTA and MgCl2. The incubation medium contained 100 fA extract and 400 ^1 of a mixture comprising 50 mM HEPES-KOH pH7.5, 10 mM KN0 3 , 0.2 mM NADH, and 10 ^M 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 [A 0.5 M zinc acetate. Excess NADH was oxidized with phenazine methosulphate (final concentration 10 ftM). Nitrite was revealed as above and NRA was expressed as nmol of nitrite min"' mg~' 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-HCI and 1% (v/v) 2-mercaptoethanol. Nucleic acids were then extracted by phenol'chloroform coupled with Reversible phosphorylation of nitrate reductase 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. 61 ATPase, cDNA from a beta subunit of Nicotiana plumbaginifolia (Boutry and Chua, 1985); TUB, cDNA from alpha tubulin of Daucus carota (Borkid and Sung, 1985). Results Northern analysis 20 /xg 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-P32] dCTP (111 TBq 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 — 80 °C using intensifying screens. Intensity of the bands was estimated after scanning and digitization using a Microtek Color/Gray scanner (Biorad) connected to a Macintosh LCIII (Apple) computer. The 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 al., 1996); GS1, a complete cDNA from cytosolic glutamine synthetase of Nicotiana tabacum (gift of B. Hirel, unpublished); In vivo NRA in isolated roots after their transfer into a liquid medium Roots were excised and immediately transferred into liquid medium. In vivo NRA decreased about 63% within the first 3 h following the transfer. Thereafter, NRA diminished slowly until the 3rd day and remained stable from the 3rd day onwards (Fig. 1). The level of NR protein, measured by ELISA analysis, remained stable during the first hours, but severely decreased 12 h after the transfer (Fig. 1). The level of total soluble proteins remained stable during the first 2 d and subsequently decreased about 20% (data not shown). Thus, a decrease of the soluble protein level could not explain the decrease of NRA during the first 2 d. In vitro NRA a 1 3 96 120 Tune (hours) Fig. 1. Time-course of in vivo NRA and NR protein levels in detopped chicory roots. Roots of 18-d-old plantlets were detopped and transferred into liquid medium. 100% activity was referred as NRA at the onset of the transfer (T = 0). Means±SD (n=10) The NR-protein level was measured by the two sites ELISA method. One representative ELISA measurement among three repeats was figured. In vitro NRA was measured in roots before they were excised. In the presence of EDTA in the reaction mixture, NRA reached 12 nmol NO2~ min" 1 mg" 1 protein. Without EDTA, but in the presence of 5 mM magnesium, it was decreased by about 60% (Table 1). 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. (1995), Mg 2 + ions promote the linkage between active phosphorylated NR and NIP (nitrate reductase inhibitor protein). EDTA chelates divalent cations and thus prevents the formation of the inactive complex. Thus, measures with EDTA reflect potential NR activity depending on the level of NR protein. With Mg 2 + ions, NRA depends on the level of active NR under either a dephosphorylated or phosphorylated form. Consequently, the ratio between NRA measured with Mg 2 + ions and NRA measured with EDTA, reflects the ratio between active NR and total NR. Three hours after the roots were transferred, the level of NR protein was not much modified (Fig. 1). Similarly, Table 1. In vitro NRA: effects of excision and addition of 0.1 nM staurosporine or I JXM okadaic acid Inhibitors were added aseptically into the liquid medium prior to the transfer of excised roots. In vitro NRA was assayed with either EDTA or Mg 2 * ions. One significant experiment is figured among three repeats. Activities were expressed as nmol NOf min" 1 mg" 1 total soluble protein. The percentage of activation corresponds to the ratio of NRA with Mg 2 + /NRA with EDTA x 100. Before excision After excision 5d 3h In vitro NRA with EDTA In vitro NRA with Mg^ + Percentage of activation 12±l 6 4.9 ±1.4 40 Control Staurosporine Okadaic acid Control 10.8 ±1.22 <1.6 <15 9.3 ±1.5 4.8±1.1 52 6.5± 1.5 nd nd 3.40 ±1.6 nd nd 62 Vuylsteker et al. NRA assayed in the presence of EDTA was not significantly reduced: it reached 10.8 nmol min~' mg" 1 protein (versus 12 nmol min" 1 mg" 1 protein before the roots were detopped). Conversely, in the presence of Mg 2 + ions NRA was inactivated over 85% (Table 1). Thus inhibition of NRA during the first hours was probably related to increased phosphorylation of NR. Five days after the transfer, NR assayed with EDTA only reached 3.40 nmol min" 1 mg" 1 protein. The level of NR protein which dropped about 66% 12 h after the transfer, remained low on day 5 (Fig. 1). Thus, whereas initial inactivation of NR during the first 3 h, was related to increased phosphorylation which was probably followed by the formation of an inactive complex. Loss of NRA on day 5 would be related to lowered levels of NR protein. Northern blot analysis Northern blot analysis did not reveal any modification of the mRNAs of either NR, GSl or jSATPase during the first hours following the transfer of the roots into the liquid medium (Fig. 2). Thereafter, theNR, GSl, tubulin, and /SATPase mRNA levels decreased by about 60% between the 3rd and the 4th days (Fig. 2) and decreased NRA may be accounted for by lowered transcription of the NR gene. However, decreased transcription was not specific to NR. Effect of inhibitors of protein phosphorylation 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 Time (h) NR 72 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 1 fiM okadaic acid to the liquid medium, strongly emphasized the in vivo NRA drop (Fig. 3). Conversely, addition of 0.1 nM staurosporine increased NRA which reached a level exceeding the initial one. Moreover, this stimulatory effect was maintained for at least 24 h (Fig. 3). For in vitro NRA assays, roots were excised and transferred into liquid medium containing either okadaic acid or staurosporine; NRA was measured 3 h later. Okadaic acid dramatically decreased NRA assayed in the presence of EDTA. As NRA was severely lowered in the presence of okadaic acid, accurate measurements of NRA could not be carried out in the presence of Mg 2 + ions. In the presence of staurosporine, NRA was more modified when assayed with Mg 2 + ions than with EDTA (Table 1). Indeed, NRA in the presence of EDTA was lowered by 17%. In the presence of Mg 2 + ions, activities were three times higher than in control excised roots attaining values measured in roots of intact plantlets. Sequential delivery of staurosporine In a first set of experiments, roots were excised, transferred for 3 h into media with or without staurosporine and assayed for in vivo NRA. It was 3.5-fold higher with staurosporine than in controls (Fig. 4a). When staurosporine was added 3 h after the roots were transferred, NRA 160 1 uM Okadaic acid Control 0,1 nM Staurosporine 96 120 ••III 80 ATPsynthase TUB GS 1 28SrRNA — HHHHH Time (hours) Fig. 2. Northern blot analysis of total RNA from excised chicory roots. The time-scale corresponds to different stages of the culture in liquid medium. Probes were cDNAs of nitrate reductase (NR), f) subunit of ATP synthase (ATP synthase), a-tubuline (TUB), and cytosolic glutamine synthetase (GSl). The lower panel corresponds to 28S rRNA coloured with acridine orange. It allows control of the loaded amounts of RNA. Fig. 3. Effect of 0.1 nM staurosporine and 1 ^M okadaic acid on in vivo NRA. Staurosporine and okadaic acid were added 3 h after the roots were transferred. NRA was expressed as nmol NOf min' 1 g"1 FW. Values are means±SD (n = 5) of one significant experiment. Assays were performed three times and the data varied within the same order of magnitude. Reversible phosphorylation of nitrate reductase 4.a 4.b 4.c Fig. 4. In vivo NRA in relation to sequential delivery of 0.1 nM staurosporine. (a) Roots were excised and transferred for 3 h into liquid medium with or without staurosporine (D) NRA in intact roots. NRA in excised roots grown for 3 h with ( • ) or without ( • ) staurosporine. (b) Excised roots harvested on control plantlets were first grown in liquid medium for 3 h. Staurosporine was then added and NRA assayed 3h latter. ( • ) NRA in roots grown with staurosporine. NRA in controls grown for 3 h ( • ) and 6 h (D) without staurosporine. (c) Roots were grown for 48 h in liquid medium before staurosporine was added and NRA was assayed 3 h later Symbols are as in (b). (d) ( • ) NRA in roots of intact plantlets incubated during 4.5 h with 0.1 nM staurosporine ( • ) controls. Roots were then harvested on plantlets that were treated for 4.5 h with staurosporine, transferred for 3 h in liquid medium and assayed for NRA. ( • ) NRA with staurosporine (GO) NRA without staurosporine. This figure represents one significant experiment from three similar repeats. assayed 3 h later was increased about 250% and recovered NR activities of undetopped roots (Fig. 4b). When the supply of staurosporine occurred 2 d after the roots were transferred, NRA was only increased by 60% and never recovered NR activities measured in intact roots (Fig. 4c). Decrease of the NR protein level (Fig. 1) probably accounts for this result. In a second set of experiments, staurosporine was supplied for 4.5 h to intact plantlets. Roots were then excised and immediately assayed for in vivo NRA which was not significantly modified compared to controls (Fig. 4d). Roots of pretreated plantlets were then excised, transferred for 3 h into liquid media with or without staurosporine and assayed for in vivo NRA. NR activities were maintained in both conditions (Fig. 4d) indicating that pretreatment with staurosporine suppressed the excision-induced drop of NRA. Discussion When excised roots of young chicory plantlets were transferred into liquid medium, NRA decreased about 60% within the first 3 h. Then, NRA decreased slowly and was stabilized from the 3rd day on. 63 NRA decreased well before the level of the NR protein which decreased about 50% 12 h after the transfer of the roots. Although the level of NRA does not always match NR protein (Oaks, 1994), the data could be explained in terms of post-translational regulations, such as reversible phosphorylation-dephosphorylation reactions of the NR protein. According to Kaiser, in vitro reactivation of phosphorylated NR is prevented by divalent cations such as magnesium and increasing phosphorylation is associated with inhibition of NRA (Kaiser and Huber, 1994). In excised roots of chicory, increased sensitivity of in vitro NRA towards Mg 2 + ions during the first hours following decapitation, is in good agreement with the assumption of the formation of a phosphorylated inactive complex. However, in vitro NRA in roots of intact plantlets was also highly sensitive to magnesium. NRA of maize roots was more susceptible to magnesium than NRA in the leaves (Merlo et al., 1995). In order to assess whether a reversible phosphorylation-dephosphorylation reaction contributed to the regulation of NRA in excised roots, inhibitors of either phosphatases or protein kinases were used. Okadaic acid, an inhibitor of PP1 and PP2A protein phosphatases, was known to prevent the reactivation of inactivated NR in leaves and roots, indicating that reactivation requires an active protein phosphatase (MacKintosh, 1992; Glaab and Kaiser, 1993; Huber et al., 1992). In detopped chicory roots, okadaic acid emphasized the loss of in vivo and in vitro NR activities. This is in agreement with both models of Kaiser and Huber (1994) and of MacKintosh et al. (1995): inhibition of phosphatases locks the reactivation process of NR and maintains the inactive NR-NIP complex, the formation of which is activated by Mg 2 + ions, at a high level. In vitro NR activities in the presence of EDTA, were assayed in extracts made from roots which grew for 3 h with okadaic acid. Even if EDTA permits dissociation of the inactive NR-NIP complex, a 3 h treatment with okadaic acid probably favoured accumulation of the inactive NIP-NR complex and may explain why NRA remained lower than in controls (Table 1). Conversely, staurosporine, an inhibitor of serinethreonine-dependent protein kinases prevented the decrease of NRA in excised roots immediately after their transfer. Delayed additions exerted poor effect, seemingly because the level of NR protein was reduced (Fig. 1). NRA in intact roots was not modified with staurosporine, indicating that the bulk NR might be in an active dephosphorylated form. Maintaining excised roots for several days in liquid media, resulted in lowering both NR and NR-mRNA levels. In chicory, such a long-term effect might progressively exhaust the pool of NR reactivable by staurosporine. Decay of the NR protein level before NR-mRNA could be accounted for by the hypothesis that phosphorylated NR is a preferential target for protein degradation (Kaiser and Huber, 1994). In addi- 64 Vuylsteker et al. tion to NR-mRNA, the levels of GS1 and jSATPase mRNAs equally decreased. The absence of growth in excised roots and suppression of shoot to root correlations probably reduced sink strength and several biosynthetic pathways such as nitrogen assimilation and respiration. Several factors may be modified by excision. First, sucrose starvation could hinder the nitrogen assimilation pathway (James et al., 1993). Secondly, suppression of the aerial part of the plants reduced the pool of available nitrate in barley roots within a few hours (Zhen et al., 1992). In detopped roots of chicory these factors probably did not account for decreased NRA, since sucrose was added to both solid and liquid media. Moreover the nitrate content was not decreased several hours after excision (data not shown) indicating that nitrate was probably not a limiting factor of NRA in excised roots. In conclusion, in young chicory plantlets, NRA, which mainly occurs in roots, is probably under metabolite control. Excision of roots which affect the metabolite balance induced a rapid inhibition of NRA. This regulation involves the phosphorylation state of the NR protein. Phosphorylation of NR can also be considered as the result of a general activation of phosphorylation reactions in response to a stress. Activation of protein kinase activities by wounding has indeed been reported (Usami et al., 1995). Acknowledgements We thank Dr G Conejero (INRA, Montpellier) for the gift of a maize anti-NR polyclonal antibody; Dr M Caboche for the gift of the maize monoclonal antibody and Dr T Moureaux (INRA, Versailles) for her helpful assistance in ELISA determination of NR contents, Dr M Boutry (University of Leuven) and Dr B Hirel (INRA, Versailles) for the generous gift of the N. plumbaginifolia j3 ATP synthase-cDNA and the N. tabacum glutamine synthetase GSl-cDNA, respectively. This work was supported by grants from Conseil Regional Nord-Pas de Calais. Christophe Vuylsteker was supported by a MERS fellowship. References Beevers L, Hageman RH. 1980. Nitrate and nitrite reduction. In: Stumpf PK, Conn EE, eds. The biochemistry of plants. New York: Academic Press, 115-68. Bachmann M, McMichael Jr RW, Huber JL, Kaiser WM, Huber SC. 1995. 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