enzymes nitrogen assimilation undergo seasonal fluctuations

Plant Physiol. (1991) 97, 322-3290032-0889/91 /97/0322/08/$01 .00/0

Received for publication November 29, 1990Accepted March 18,1991

Enzymes of Nitrogen Assimilation Undergo SeasonalFluctuations in the Roots of the Persistent Weedy Perennial

Cichorium intybus'

Konrad A. Sechley*, Ann Oaks, and J. Derek BewleyDepartment of Botany, University of Guelph, Guelph, Ontario, Canada, NlG 2W1

ABSTRACT

Chicory (Cichorium intybus), a deep rooted weed, grows inregions with temperate climates. Seasonal partitioning of com-pounds between the root and shoot results in fluctuations in thesoluble carbohydrate, nitrate, amino acid, and protein pools withinthe roots. The activities of nitrate reductase (NR) (EC 1.6.6.1),glutamine synthetase (EC 6.3.1.2), NADH (EC 1.4.1.14), ferrodoxinglutamate synthase (EC 1.4.7.1), and glutamate dehydrogenase(GDH) (EC 1.4.1.2-4) vary throughout the year and coincide withseasonal alterations in nitrate, fructose, and sucrose. During thewinter, NR, glutamine synthetase and ferrodoxin glutamate syn-thase activities increase in the root, while GDH displays theopposite trend with elevated activity in the summer months. Allof these enzymes exhibit seasonal alterations in abundance asdetected by Western blot analysis, increasing during the winterand, therefore, contributing to the seasonally dynamic proteinpool. Extensive fluctuations in abundance and activity of theseenzymes in the root occur during the spring and fall and coincidewith shoot growth and senescence, respectively. Several obser-vations indicate that posttranslational modifications of NR andGDH are taking place throughout the year; for example, NR isparticularly unstable during the spring and fall, and seasonal GDHactivity does not correlate with protein abundance.

The persistence of perennials in regions of temperate cli-mates involves seasonal cycling of nutrients between shootsand roots, because aerial portions ofthese plants die offduringwinter. Export of photosynthate from leaves and stems to theroots in the fall, and their remobilization from subterraneanorgans to facilitate regrowth in the spring, is presumablytightly regulated. Yet little is known about either the biochem-ical processes involved or how environmental cues are per-ceived and interpreted by roots in a seasonal manner.

Vegetative storage proteins accumulate in leaves (25) androots (13) of soybean in response to sink demand, wounding,and water stress, in tuberous perennials such as artichoke (15)and potato (19), and in overwintering roots ofperennial weeds(3). In roots of chicory (Cichorium intybus), dandelion, andleafy spurge, total proteins increase on a dry weight basisduring the winter months (2, 4), which may be an adaptivestrategy for freezing tolerance. Nitrate and total amino acids

Research supported by the Natural Sciences and EngineeringResearch Council ofCanada (NSERC), grant A22 10 to J.D.B., A28 18to A.O.. and an NSERC Postdoctoral Fellowship to K.A.S.

also increase in chicory roots in winter, with glutamine,asparagine, serine, ornithine, and glutamate together account-ing for 65 to 70% of the soluble amino acid pool (4). Theconcomitant increases in soluble protein, nitrate, and aminoacids in overwintering roots is suggestive ofsignificant changesin activity of the enzymes responsible for nitrogen assimila-tion. Here we demonstrate that, indeed, this is the situationin chicory roots, and that enzyme activity and amount exhibitmarked seasonal fluctuations to account for the changes incomponents of the nitrogen pool.

MATERIALS AND METHODS

Chicory (Cichorium intybus L.) was grown in the weedgarden at the University of Guelph under natural conditionswith no external sources of nutrient. Roots were collected ona monthly basis from five plants per harvest as outlined byCyr and Bewley (2) during 1987/1988. The samples werelyophilized and stored as powders at -20°C. Enzyme assayswere performed on material obtained from three separateplants obtained at each of these time points. To determinethe stability of enzymes within these samples, the activities ofNR2, GS, and GDH were compared with samples obtainedin November and July during the 1989/1990 season. Thevariation between plants during both seasons (up to 18%) wassimilar to the variation observed between seasons (up to 21 %).

Nitrate Reductase, Glutamate Dehydrogenase, NADHGlutamate Synthase, and Fd-Glutamate Synthase

Proteins from the lyophilized powders were solubilized(10:1 [v/w] lyophilized material) at 4°C in 50 mM Tris-HCl,pH 7.5, 12.2 mm f-mercaptoethanol, 10 mM PMSF (Sigma),and polyvinylpolypyrrolidone (1:1 [w/w] lyophilized mate-rial). The extraction buffer contained 1 mM EDTA whensamples were to be assayed for Fd-GOGAT. The slurry wasplaced on ice for 15 min with intermittent vortexing prior tocentrifugation at 10,000g for 10 min, and the supernatantdesalted through Sephadex G25 (Pharmacia LKB Biotech-nology, Baie D'Urte, Quebec).For determination of enzyme activity, reaction mixtures

contained 0.1 to 0.25 mg protein (Bio-Rad protein assay,Mississauga, Ontario), over which range the assays were linear.NR (EC 1.6.6.1) assay mixtures containing extracted pro-

2 Abbreviations: NR, nitrate reductase; GDH. glutamate dehydro-genase; GOGAT, glutamate synthase; GS, glutamine synthetase.

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SEASONAL FLUCTUATIONS OF NITROGEN ASSIMILATING ENZYMES

tein, 10 mM KNO3, and 0.2 mM NADH in a total volume of1.0 mL, were incubated at 25°C for 30 min before terminationwith 0.1 mL of 0.1 M zinc acetate and centrifugation at1 6,000g for 3 min. Production of nitrite was determined withsulfanilamide and N-( 1-napthyl)ethylenediamine dihydro-chloride at 540 nm (5).GDH (EC 1.4.1.2-4) assay mixtures contained extracted

protein, 150 mm ammonium acetate, 12.4 mm 2-oxo-glutar-ate, and 1 mm CaCl. The reaction was initiated with 80 AMNADH, and the oxidation of NADH was determined at 340nm over 10 min at 25°C (l1).NADH-GOGAT (EC 1.4.1.14) assay mixtures contained

extracted protein, 5 mm glutamine, 5 mm oxo-glutarate, and80 AM NADH, and NADH oxidation was determined as forGDH (1 1). For the assay of Fd-GOGAT (EC 1.4.7.1), methylviologen, final concentration 20 mm, was added in place ofNADH. The reaction was initiated with Na2S204 and Na-HCO3, both at a final concentration of 7 mg mL-', andincubated for 20 min at 30°C. Glutamate, the product of thereaction, was separated from the rest of the mixture by Dowex1 (acetate form), incubated for 10 min at 80°C with ninhydrin,and its concentration determined at A506 (8, 11). Activitieswere corrected for glutamate produced in assays that did notcontain any reductant (Na2S2O4 and NaHCO).

Glutamine Synthetase

Proteins were extracted and desalted as outlined above in50 mM Tris-HCI, pH 8.0, 1 mM EDTA, 1 mM DTT, and 10mM Mg2SO4. GS (EC 6.3.1.2) activity was determined biosyn-thetically in mixtures containing 0.1 to 0.25 mg protein, 50mM Tricine-KOH, pH 7.8, 80 mm glutamate, 20 mM Mg2SO4,4 mm EDTA, 6 mm hydroxylamine, and 8 mM ATP, andincubated for 10 min at 37°C. The reaction was terminatedwith an equal volume of FeCl3 reagent (0.37 M FeCl3 in 0.67M HCl and 0.2 M TCA), the samples were centrifuged at1 6,000g for 3 min, and the production of glutamyl hydroxa-mate was determined at 540 nm (18).

Gel Electrophoresis and Western Analysis

Tissue proteins were solubilized (10:1 [v/w] lyophilizedmaterial) in 0.1 M Tris-HCl, pH 7.5, 10 mM cysteine, 10 mMPMSF, and polyvinylpolypyrrolidone (1:1 [w/w] lyophilizedmaterial) on ice for 15 min. Centrifugation was at 10,000gfor 10 min, followed by acetone precipitation, drying, andsolubilizing in 0.2 M Tris-HCl, pH 6.8. The protein concen-tration was determined, and the extract was brought to a finalconcentration of 0.19 M Tris-HCl, pH 6.8, 2% (w/v) SDS,and 5% (v/v) f3-mercaptoethanol (sample buffer). Samplescontaining 10 jg of heat-denatured protein were separated byone dimensional SDS-PAGE (8 or 12.5% [w/v] acrylamidegels) and stained with Coomassie brilliant blue R-250. ForWestern analysis, the sample buffer was filtered through ni-trocellulose. This reduced the nonspecific binding betweenthe antisera of NR, GOGAT, and GDH and (unidentified)components of the buffer. Gels were electroblotted at 100 Vfor 1 h onto nitrocellulose (Bio-Rad) and developed in 10 mMTris-HCl, pH 8.0, 0.15 M NaCl, and 0.05% (v/v) Tween 20.Dried milk (5% [w/v]) was used as the blocking agent. Blotting

efficiency was determined by restaining the polyacrylamidegels and by monitoring the extent of transfer of rainbow molwt markers (Amersham, Oakville, Ontario). Antibodies wereused at the following dilutions: NR (12) 1:600; GDH (10)1:500; Fd-GOGAT (27) 1:3000; and GS, and GS2 (9) 1:750.Because the results with GS, and GS2 antisera were similar,due to a high degree of sequence similarity between these twoisozymes (6), only those with GS2 antisera are presented.Relative amounts of polypeptides were determined by scan-ning Western blots with a GS300 transmittance/reflectancescanning densitometer (Hoefer Scientific Instruments, SanFrancisco, CA) attached to an integrator and set to the reflec-tance mode.

Two-Dimensional PAGE

Acetone-precipitated samples containing 15 Mug of proteinwere solubilized in 1.5 M Tris-HCl, pH 8.9, and 50% glyceroland separated on native gels (10% [w/v] acrylamide). Thesegels were then equilibrated in 0.19 M Tris-HCl, pH 6.8, 2%SDS (v/v), and 5% ,3-mercaptoethanol (v/v) for 20 min, andproteins were separated on 12.5% (w/v) acrylamide 1% SDSgels in the second dimension, followed by electroblotting ontonitrocellulose (as above), or staining with Coomassie brilliantblue R-250.

Activity Staining

To determine GS activity, samples were separated by nativePAGE (10% [w/v] acrylamide). The gel was rinsed in 50 mMTricine-KOH, pH 7.8, 80 mm glutamate, 20 mM Mg2SO4, 4mM EDTA, and 6 mm hydroxylamine for approximately 1min prior to overlaying the gels with fresh reagents including8 mm ATP, for the assay of GS. For determination of phos-phate, one ofthe products ofthe reaction, the assay contained4 mM NH4Cl in place of hydroxylamine. Gels were incubatedfor 15 min at 35°C before the solution was aspirated off.Phosphate was determined (18) by overlayering the gels witha mixture containing 4 mL FeSO4 reagent (0.8 g FeSO4. 7H2Oin 10 mL 0.15 N H2SO4) and 3 mL ammonium molybdatereagent (0.66 g ammonium molybdate in 10 mL 7.5 N H2SO4)for 2 min at 20°C. In a separate set of gels, the production ofglutamyl hydroxamate (synthesized in the presence of hy-droxylamine) was detected with the addition of FeCl3 reagentused for in vitro GS activity.For the detection of GDH activity, samples were extracted

in 100 mm Tris-HCl, pH 8.0, 1 mm DTT, and 1 mm CaCl2as outlined above, and proteins in an aliquot of the superna-tant were separated by native PAGE (10% [w/v] acrylamide)at 4°C. The gels were equilibrated in the same buffer withoutDTT for 5 min and overlayered with fresh reagents for theassay ofGDH activity (deaminating) by detecting the produc-tion of NADH. This buffer consisted of 100 mm Tris-HCl,pH 8.0, 1 mm CaCl2, 1 mm NAD+, 50 mm glutamate, 0.43mM nitroblue tetrazolium, and 0.05 mg mL-' of phenazinemethosulfate. The gels were incubated for 30 min at 35°C inthe dark with occasional agitation.

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RESULTS AND DISCUSSION

Changes in the metabolic constituents of chicory roots area result of many interacting environmental stimuli that theplant (and root) experience throughout the year. Seasonalpartitioning of reserves, cold acclimation, and changes innutritional supply and photoperiod lead to fluctuations in thepools of fructan, nitrate, amino acids, and proteins (4). Theaccumulation of a particular protein within a storage organdoes not necessarily imply that it has a storage role, e.g.patatin, a major storage protein that accumulates within thetubers of potato, exhibits lipid acyl hydrolase activity (21).Thus, the observed seasonal alterations in the protein pool inchicory roots (3, 4) may include quantitative changes inphysiologically active enzymes.

Nitrate Reductase

The assimilation of nitrate in plants to produce ammoniuminvolves NR and nitrite reductase at the expense of NADH,NADPH, and/or reduced Fd. NR activity is substrate-induc-ible (17) and also increases when roots are incubated inglucose, sucrose, or fructose ( 16, 22). Because nitrate, sucrose,and fructose increase in chicory roots during the winter (4),we initially examined whether this enzyme exhibits parallelchanges in activity to its substrate and to solublecarbohydrates.A pronounced seasonality of NR activity is evident when

expressed on either a protein or a dry weight basis (Fig. 1A).Increased root NR activity is apparent during the late fall andwinter months and thus precedes the increase in nitrate,sucrose, and fructose (4). Hence, the storage pools of thesecompounds appear not to play a direct role in regulating NR.A lack of correlation between potential inducing substancesand enzyme also occurs in the spring, when NR activities arehigh even though nitrate levels within the root are declining.The metabolic pool of nitrate (versus the storage pool withinthe vacuole) has been implicated in the regulation ofNR (7),and this source may play a role in mediating NR activityduring the fall and spring. A reduced demand for nitrate inthe fall in the shoots, and its remobilization for new shootgrowth in the spring, could lead to transient alterations in thesupply through the vascular system, thereby varying its fluxthrough the metabolic pool. There is no NR activity duringthe summer months when the pool of nitrate within the tissueis low.

It is not known whether chicory preferentially exports ni-trate to the shoot or reduces nitrate within the root. Becausethe extractable activities ofNR are lower than expected for a

plant that reduces nitrogen within the root, e.g. Pisum (1, 26),it seems plausible that the aerial portions of chicory are

responsible for the majority of the reduction of nitrate duringthe summer. Shoot nitrate reduction does occur in otherperennial species, e.g. Capsicum, Gossypium, or Impatiens(1). Within these species, the availability of soil nitrate affectsthe partitioning of nitrate reduction between the root andshoot, with the shoot becoming important as the externalnitrate concentration increases (1). The increase in NR activ-ity in the root during the winter period may, therefore, be inresponse to seasonal fluctuations in the availability of soil

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Figure 1. Seasonal patterns of NADH-NR protein and activity inchicory roots. A, NR activity in root extracts prepared in the presenceof PMSF and expressed on a total protein (A), and on a dry weight(l) basis. Data are means ± SE of three replicates. B, Western blotof root polypeptides exposed to NR antiserum. Root extracts wereseparated by SDS-PAGE (8% [w/v] acrylamide), transferred, andprobed with NR antiserum (12). The solid arrowheads indicate NR(116 and 38 kD), and the open arrowhead indicates nonspecificbinding of the antiserum (see "Materials and Methods"). Mr, relativemolecular mass in kD.

nitrate coupled with defoliation and the loss of the capacityfor nitrate reduction in the shoot.The persistence ofNR during the spring months may occur

because NR is not subjected to proteolysis, or it is differen-tially stabilized at this time. Because NR-specific proteasesfrom roots of corn have been characterized (30), we deter-mined the stability of NR in crude extracts of roots madethroughout the year. For these experiments, samples wereprepared in the presence or absence of PMSF, an inhibitor ofproteolytic activity. Similar NR activities are present in ex-tracts of all monthly samples isolated with or without theinclusion of PMSF (Fig. 2A and B). When extracts lackingPMSF are incubated on ice for 3 h before assaying for NR,its activity is reduced approximately twofold in the fall andwinter samples, and is absent from the spring samples, a

period when normally there is enzyme activity. However, asimilar loss ofactivity occurs in samples prepared with PMSF,indicating that reduced activity in vitro may be due to eithera PMSF-insensitive protease, or an inherent instability ofprotein following extraction, this being more evident in ex-

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glutamate catalyzed by GS (17). The supply of glutamate asa substrate for GS, as well as for other metabolic processes, isprincipally provided through the activity of GOGAT; thecombined activities are considered to operate as a GS-GO-GAT cycle.GS activity is detectable throughout the year in root extracts

(Fig. 3A). Activity is high from the late fall to late winter,while spring and summer activities decline 25-fold, expressedon a dry weight basis, or sixfold when expressed on a totalprotein basis. The decrease in activity during the summer isnot due to the presence of soluble components within thesesamples. Co-extracted tissues (on a dry weight basis) harvestedfrom June and February exhibited an average value of GSactivity (72 nmol glutamyl hydroxamate min' mg protein-'),compared with activities in February and June of 108 and 27nmol glutamyl hydroxamate min' mg protein-', respectively.Even though GS activity increases in roots incubated withnitrate (29), sucrose, glucose, or fructose (22), elevated GSactivity in chicory roots precedes a rise in these pools in thefall, while in the spring nitrate declines prior to a drop in GSactivity. As with NR activity, this indicates that these com-

Month of year

Figure 2. Stability of NR activity in the presence and absence ofPMSF. Extracts were prepared with (A) or without (B) 10 mM PMSFand assayed for NR activity immediately (A, A), or after 3 h incubationon ice (V, V). Data are means ± SE of three replicates.

tracts from the spring and early fall. Furthermore, the decreasein activity during the summer period is not a result of a

soluble inhibitor ofNR activity present at this time. Samplesharvested in June and February were co-extracted (on an

equal dry weight basis), and the resulting activity (0.031 nmolNO2- min' mg protein-') was an average of the June (notdetectable) or February (0.057 nmol NO2- min-' mg pro-tein-') activities.

Western blots incubated with NR antiserum reveal threebands of M, 116,000, 62,000, and 38,000. The 116 and 38kD bands increase approximately 10-fold during the wintermonths (Fig. 1 B) and are absent in the summer. These bandsare not detected when the NR antiserum is incubated with a

protein extract from maize containing NR protein prior toincubation with the Western blot, yet they bind NR antiserumpreincubated with a maize protein extract lacking NR protein(data not presented); this was not observed for the 60 kDband (see "Materials and Methods"). This indicates that theI 16 and 38 kD polypeptides are representative ofNR protein.Because the in vitro assays demonstrate an inherent instabilityofactivity, the 38 kD polypeptide may represent a degradationproduct derived from the 116 kD polypeptide. The lack ofcorrelation between NR activity and protein during the springfurther suggests that NR activity is regulated, or that theprotein is turning over rapidly during the period ofnew springgrowth.

Glutamine Synthetase

The major point of entry ofammonium into an organicallycombined form in plant tissues involves the amination of

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Figure 3. Seasonal patterns of GS protein and activity in chicoryroots. A, GS activity expressed on a total protein (A), and on a dryweight (L) basis. Data represent means + SE of three replicates. B,

Western blot of root proteins separated by SDS-PAGE (12.5% [w/v]acrylamide), transferred, and exposed to GS2 antiserum (9). Closed

arrowheads indicate GS1 (39 kD) and GS2 (42 kD) polypeptides; the

open arrowheads point to 51 and 72 kD polypeptides that also bind

the antiserum. Similar results were obtained when blots were ex-

posed to GS, antibody. Mr, relative molecular mass in kD.

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ponents of the nitrogen and carbohydrate storage (vacuolar)pools do not play a role in the regulation of GS activity.GS activity in vitro is due to both the cytosolic and the

plastid isozymes, GS1 (M, 38,000-41,000) and GS, (Mr41,000-45,000), respectively (6, 9). The occurrence of GS, inroot tissues has been reported by some workers (14, 29), butnot by others (26, 28). This discrepancy may represent eithercompositional differences between species (as is the case forleaf GS, and GS2) (6), or altered developmental responses toenvironmental factors such as nutrient supply, as GS, hasbeen detected in roots of pea growing under steady-stateconditions in the presence of nitrate (29). Therefore, wedetermined whether or not the increase in GS activity in theroots during the winter is a result ofdifferences in the isozymespresent.GS activity, detected after separation by nondenaturing

PAGE, is present during the fall and winter months but absentduring the summer (data not presented). To permit charac-terization of the GS isozymes by Mr, extracts of roots har-vested in February and June were separated on nondenaturinggels in the first dimension followed by SDS-PAGE in thesecond. Western blot analysis of the separated proteins revealsa band from the February roots with GS activity that migratesto 42 kD (GS,) (Fig. 4A). The GS antiserum also binds to two39 kD polypeptides (GS,), present in different amounts, thatare separated during the first dimension on nondenaturingPAGE. Two other polypeptides (51 and 72 kD) are alsoevident in the extracts of February roots. In the June extract(Fig. 4B), which exhibits no GS activity in nondenaturinggels, the 42 kD polypeptide is barely detectable in Westernblots, and one of the 39 kD polypeptides is in much lowerproportions than in the February roots. The 51 kD polypep-tide is lower also, and the 72 kD polypeptide is absent (Fig.4B).

Seasonal fluctuations in GS protein and isozyme composi-tion were further characterized by Western analysis (Fig. 3B).The cytosolic GS, (39 kD) form is present in the rootsthroughout the year, but it is up to twofold higher during thewinter than in the summer. This increase is reflected in oneof the polypeptides of the 39 kD doublet, discerned by two-dimensional PAGE (Fig. 4). On the other hand, GS, (42 kD)is six- to sevenfold lower in roots during the summer monthswhen compared to those from October through March (Fig.3B). The sixfold variation in total GS activity in vitro (ex-pressed on a total protein basis) (Fig. 3A) correlates well withsimilar changes in the GS, isozyme (Fig. 3B; as shown bydensitometry of samples loaded on an equal total proteinbasis). There are seasonal variations in the 51 and 72 kDpolypeptides also, with those of the latter resembling thevariations in GS,.

Glutamate Synthase

GOGAT catalyzes the transamidation of glutamine to oxo-glutarate, producing two moles of glutamate at the expenseof either NAD(P)H or Fd. Both forms ofGOGAT have beenreported in roots ( 17). We examined the seasonal changes inNADH-GOGAT and Fd-GOGAT, although the activity ofonly the former is correlated with nitrate supply (8, 23).A marked seasonal variation in NADH-GOGAT activity is

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Figure 4. Western blots of proteins separated by two-dimensionalPAGE and exposed to glutamine synthetase (GS2) antiserum. Chicoryroot proteins obtained in February (A) and June (B) were separatedby non-denaturing PAGE in the first dimension and in the presenceof SDS in the second dimension, transferred, and probed with GS2antiserum (9). Open arrowheads indicate GS2 (42 kD), and the closedarrowheads point to the one 39 kD polypeptide (GS1) that differs inabundance between the two samples. Similar results were obtainedwith GS, antibody. M,, relative molecular mass in kD.

evident, it being 200-fold lower (on a dry weight basis, and250-fold on a protein basis) in summer than in winter (Fig.5). Alterations are also apparent in Fd-GOGAT activity,varying fivefold or 20-fold, on a protein or dry weight basis,respectively (Fig. 6A). Based on co-extraction experiments,there was no evidence for a soluble inhibitor of GOGATactivity in summer samples. Fd-GOGAT activity is 20-foldhigher than NADH-GOGAT and approaches the levels ofGSactivity (Fig. 3A). Both NADH- and Fd-GOGAT activitiesparallel those ofGS (and NR, Fig. IA), but only GS and Fd-GOGAT activities are similar in their degree of variation.NADH- and Fd-GOGAT activities closely follow the seasonal

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Figure 5. Seasonal patterns of NADH-GOGAT activity in chicoryroots. NADH-GOGAT activity expressed on a protein (A) and on adry weight (L) basis. Data are means ± SE of three replicates.

changes in nitrate, aside from an increase in September thatprecedes the increase in nitrate.A direct comparison between NADH-GOGAT activity and

NADH-GOGAT protein cannot be made because, to the bestof our knowledge, no antibody is available. Therefore, weexamined the seasonal fluctuations in Fd-GOGAT protein.Fd-GOGAT protein (172 kD), as detected by Western blotanalysis of extracts separated by SDS-PAGE, is presentthroughout the year and is fivefold higher in roots during thewinter months than in the summer (Fig. 6B). The fivefoldalteration in Fd-GOGAT activity correlates well with thechanges in Fd-GOGAT activity (expressed on a protein basis).The fluctuations in Fd-GOGAT protein and activity (fivefold)approximate those of GS, protein and activity (six- to seven-fold). Increased Fd-GOGAT protein (i.e. threefold higher thanin mid-summer) is still present in April, yet extractable Fd-GOGAT activity is lower at this time. Conversely, higher Fd-GOGAT activity is detected early in the fall prior to maxi-mum Fd-GOGAT protein in October.The seasonal variation in Fd-GOGAT protein and activity

in chicory roots is interesting because the activity of thisenzyme is apparently not responsive to nitrate (8). Yet, inmaize we have observed increased Fd-GOGAT activity in thepresence of nitrate (our unpublished data). However, thepossibility exists that other environmental signals may also beresponsible for its fluctuations.

Glutamate Dehydrogenase

During the summer months, when NADH-GOGAT activ-ity is barely detectable, glutamate (a product of GOGATactivity) might be in short supply for ammonium assimilationby GS or other metabolic processes within the root. BecauseGDH may be involved in glutamate production and ammo-nium assimilation (17, 24), and because it is also sensitive tocold (24) or the addition of fructose or sucrose (22), weexamined whether or not activity ofGDH in the roots changesduring the year.A yearly fluctuation in NADH-GDH activity is apparent

(Fig. 7A), with levels being 2.5-fold higher in the summerthan in the winter when expressed on a protein basis, yetactivity decreases in the fall when expressed on a dry weightbasis. GDH activity typically responds in an inverse mannerto GS (20, 22, 24), and this relationship is observed withinchicory roots when calculated on a protein basis.

Separation of proteins by nondenaturing electrophoresisand assaying for GDH activity reveals two bands of activity.When further separated in a second dimension by SDS-PAGEand probed with GDH antisera, these bands correspond totwo polypeptides with Mrs of 36,000 and 32,000 (data notpresented). An 84 kD band present in the SDS-PAGE dimen-sion is a contaminant of the sample buffer (see "Materialsand Methods") and appears across the width of the seconddimension. Of interest is the variation in GDH polypeptides(36 and 32 kD), as detected by Western analysis (Fig. 7B).GDH protein is present during the fall and winter months (atime when in vitro GDH activity is lowest) and parallels theseasonal trends observed for NR, GS, and GOGAT proteins.Increased protein does not implicate a parallel rise in activityas evidenced by the opposite seasonal trends in GDH proteinand activity; rather, it is possible that a reversible inactivation

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Figure 6. Seasonal patterns of Fd-GOGAT protein and activity inchicory roots. A, Fd-GOGAT activity expressed on a protein (A) andon a dry weight (El) basis. Data are means ± SE of three replicates.B, Western blot of root proteins separated by SDS-PAGE (8% [w/v]acrylamide), transferred, and exposed to Fd-GOGAT antiserum (27).The solid arrowhead points to GOGAT (172 kD), and the openarrowheads indicate two major polypeptides (50 and 56 kD) associ-ating with the antiserum but not exhibiting a seasonal trend. Mr,relative molecular mass in kD.

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of the active enzyme occurs. A lack of correlation betweenamount of GDH protein and activity has been noted also insenescing rice leaves (10) and in maize (A. Oaks, unpublisheddata). Decreased GDH activity and increased protein abun-dance during the late fall precede the increase in sucrose orfructose, suggesting that storage pools of carbohydrate are notdirectly involved in regulating GDH. Also apparent in theseblots is an 84 kD polypeptide that weakly binds the GDHantiserum and is present during the winter.

CONCLUSION

The soluble protein pool, as discerned by one-dimensionalSDS-PAGE, undergoes seasonal changes in several polypep-tides, including a partially characterized abundant storageprotein (18 kD) that increases during the winter (3) (Fig. 8).Seasonal fluctuations in the abundance of protein, as detectedby Western blots, occur in NR (1 16 and 38 kD), GS, (39 kD)and GS, (42 kD), Fd-GOGAT (172 kD), and GDH (36 and32 kD), and these contribute to the observed changes in theprotein pool (Fig. 8). Other polypeptides also change, includ-ing those detected by nonspecific binding to GS (51 and 72

Figure 8. Seasonal changes in polypeptide composition of chicoryroots. Soluble extracts of proteins from roots collected on a monthlybasis were acetone-precipitated and 10 mg protein (per lane) sepa-rated on 12.5% (w/v) polyacrylamide gels in the presence of SDS.Bars point to NR (116 and 38 kD), GS1 (39 kD), GS2 (42 kD), Fd-GOGAT (172 kD), GDH (36 and 32 kD), and a partially characterizedabundant storage polypeptide (18 kD). Mr, relative molecular mass inkD.

kD), and GDH (84 kD) antisera. Alterations within the poly-peptide pool are most evident between April and May andbetween August and September, and these transition periodscoincide with reserve mobilization during shoot emergence inthe spring and the fall preparation of aerial senescence. It isalso during these periods that extensive changes in enzymeactivity and abundance ofNR, GS, NADH- and Fd-GOGAT,and GDH take place.

Environmental stimuli responsible for the seasonal altera-tions in protein and enzyme activities are unknown. Most ofthe enzymes examined in this study are known to respond tothe nitrogen and carbohydrate status ofthe plant, componentsthat vary considerably within the chicory root (3, 4). Modifi-cation of these components of the carbon and nitrogen poolmay result from reduced sink demand elsewhere in the plantdue to seasonal defoliation. Further studies involving manip-ulation of environmental signals (e.g. temperature and daylength) are required to understand their perception within theplant, and root, on a seasonal basis.

ACKNOWLEDGMENTS

We would like to thank B. Hirel and A. Suzuki for the use of GS,&(9) and GOGAT (27) antibodies. respectively, and S. MacIsaac. T.Reynolds. and an anonymous reviewer for their useful suggestionson the paper.

Figure 7. Seasonal pattern of GDH protein and activity in chicoryroots. A, GDH activity expressed on a protein (A), and dry weight (O)basis. Data represent means ± SE of three replicates. B, Westernblot of root proteins separated by SDS-PAGE (12.5% [w/v] acryl-amide), transferred, and exposed to GDH antiserum (10). Solid arrow-heads indicate GDH polypeptides (32 and 36 kD); the open arrowheadindicates nonspecific binding of the antiserum (see "Materials andMethods"). Mr, relative molecular mass in kD.

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enzymes nitrogen assimilation undergo seasonal fluctuations

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