Plant Physiol. (1986) 81, 356-3600032-0889/86/8 1/0356/05/$0 1.00/0

Ammonia Fixation via Glutamine Synthetase and GlutamateSynthase in the CAM Plant Cissus quadrangularis L.'

Received for publication August 22, 1985 and in revised form December 8, 1985

MICHAEL G. BERGER*, MICHAEL L. SPRENGART, MISRI KUSNAN, AND HEINRICH P. FOCKUniversitat Kaiserslautern, Fachbereich Biologie, Postfach 3049,6750 Kaiserslautern, Federal Republic ofGermany

ABSTRACT

Succulent stems of Cissns quadrawgularis L. (Vitaceae) contain glu-tamine synthetase, glutamate synthase, and glutamate dehydrogenase.The CO2 and water gas exchanges of detached internodes were typicalfor Crassulacean acid metabolism plants. During three physiologcalphases, e.g. in the dark, in the early illumination period after stomataclosure, and during the late light phase with the stomata wide open,15NH4CI was injected into the central pith of stem sections. The kineticsof 15N labeling in glutamate and glutamine suggested that glutaminesynthetase was involved in the initial ammonia fixation. In the presenceof methionine sulfoximine, an inhibitor of glutamine synthetase, theincorporation of 'IN derived from 5NH4Cl was almost completely inhib-ited. Injections of amido-'5N glutamine demonstrated a potential for 'INtransfer from the amido group of glutamine into glutamate which wassuppressed by the glutamate synthase inhibitor, azaserine. The evidenceindicates that glutamine synthetase and glutamate synthase could assim-ilate ammonia and cycle nitrogen during all phases of Crassulacean acidmetabolism.

In a variety of C3 and C4 photosynthetic tissues it is widelyrecognized that ammonia assimilation proceeds via GS2 andGOGAT (1-3, 9, 28, 29). Recently, evidence has accumulatedindicating an involvement of GDH in nitrogen fixation duringplant growth at ample nitrate supply (19) or in media containingabundant ammonia (4, 21). Under simulated in vivo conditionsfavoring activity of GDH, the only mitochondrial enzyme withthe potential for ammonia assimilation (26), glutamate wasformed from ammonia derived from glycine (26) or appliedexogenously (30) in isolated mitochondria. Others suggested thatGDH may be operative at elevated intracellular ammoniumlevels or during energy limitations opposed to GS activity (16).There are a few reports on ammonium assimilation in CAM

plants. The cytoplasmic and chloroplastic isoenzymes ofGS weredetected in leaves ofboth Kalanchoe blossfeldiana and Kalanchoedaigremontiana (15). Chang et al. (5) demonstrated NADH andNADPH dependent GDH activities in leaves of Kalanchoefedt-schenkoi, the combined activities of which were similar to thefixation capacity determined for GS and higher than the in vitroactivity of GOGAT in these tissues. These authors concludedthat in leaves of K. fedtschenkoi NH3 could be assimilated by

' Financial support by the Deutsche Forschungsgemeinschaft is grate-fully acknowledged.

2Abbreviations: GS, glutamine synthetase; GOGAT, glutamate syn-thase; GDH, glutamate dehydrogenase; MV, methyl viologen; MSO,methionine sulfoximine; AZA, azaserine; fw, fresh weight.

both the GDH and the GS/GOGAT pathway.This study examines the fixation ofammonia in the photosyn-

thetic tissue of the CAM plant, Cissus quadrangularis. Thissucculent was used here, since its diurnal fluctations in theorganic acid content and its CO2 exchange patterns were typicalfor CAM succulents in the green internodes of both droughtstressed and well watered plants (24).

MATERIALS AND METHODS

Plant Material. Cuttings of Cissus quadrangularis were oKtained from a plant raised in the Botanic Gardens at the Univer-sity of Kaiserslautern. In trays (0.5 x 0.3 x 0.05 m) containingsandy loam soil cuttings were cultivated for 8 months in aphytochamber. The rooted plants were watered twice a week andwere given additionally 0.75 L nutrient solution (6). During thedaily light period (9.00-23.00 h) the RH was 60% saturation at27C. The light source consisted of fluorescent tubes (L 58 w/21, NL 65 w/30 Fluora L 58 w/77, Osram D-1000 Berlin), thephoton fluence rate at stem surface was 150 to 200 Amol/m2.sPAR. During the dark period the humidity was regulated to 90%saturation at 16°C.To avoid infestation by fungus, the plants were watered twice

(0.75 L per tray) with orthocid 50 (3 g captan/L) and also sprayedwith the fungicide about 8 weeks prior to use.CO2 and H20 Gas Exchange Measurements. An open gas

exchange system (2) was modified to simultaneously measure 3samples. The CO2 concentration of the measuring gas was ad-justed by mixing CO2 free air with CO2 using mass flow control-lers (FC 2600, Tylan GmbH, D-8057 Eching). The gas streamwas divided. The reference gas was dried and passed through thereference side of the CO2 gas analyzer (22 L/h). Internodes wereenclosed in a tube shaped plexiglass cuvette (length 300 mm,internal diameter 32 mm). Four tubes were embedded in a closedwater bath (about 10 mm water layer over the tubes) to controlcuvette temperature. Each tube was flushed with 60 L/h ofhumidifed air (100% RH at 12.5C). The gas flow rates in theindividual cuvettes were measured by flowmeters (Platon Flow-bits GmbH, D-6903 Neckargemund and Rota D-7012 Oelflin-gen). Constant pressure conditions enabling constant gas fluxesthrough the cuvettes were obtained by bubbling excess gas intowater (water column about 50 mm) using magnetic three wayvalves. The gas stream of one cuvette was led every 10 min tothe humidity sensor (Vaisala, Driesen und Kern, D-2000 Ham-burg-Tangstedt) and dried over CaCI2 and Mg(Cl04)2 beforemeasuring the CO2 concentration with an IR gas analyzer (Unor2, Maihak, D-2000 Hamburg). The temperature of the plantmaterial was measured using two cooper constantan wires at-tached to the lower surfaces of two stem sections.The rates of CO2 uptake and evaporation were calculated as

described previously (2).Experimental. For the incubations leafless sections (3.5-5 cm

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AMMONIA FIXATION IN CISSUS QUADRANGULARIS

length, 1-1.8 g fw) of mature internodes, carefully selected foruniformity, were detached from the plants. With a sharp metaltube (2 mm inside diameter) a small hole was punched throughthe central pith of the internode. After sealing one cut surfacewith silicon rubber (Teroson GmbH, D-6900 Heidelberg), theincubation solution (50 ;Ll/g fw) was injected into the hole. Afterthe other cut surface was sealed, the internode was incubated ona rotary shaker (l/s) at 0 or 200 umol/m2-s PAR under theenvironmental conditions applied during growth. Concomi-tantly, the CO2 and H20 gas exchange of equally treated stemsections was measured in an open gas exchange system. NH4Cl,glutamine, and inhibitor solutions were injected in the dark(phase I; 10) from 2.30 to 4.45 h as well as in the light between10.00 to 12.15 h (phase III, stomata closed), or between 20.45 to23.00 h (phase IV, stomata open). After the incubations (up to135 min) the rubber was removed and the internodes wereimmediately weighed. The stems were ground in a mortar inliquid N2 with 0.1 g acid washed sand (Fluka AG, CH-9470Buchs) and diatomaceous soil (Celite 545, Karl Roth KG, D-7500 Karlsruhe). During thawing, 10 ml of 80% (v/v) ethanol(pH 3 with HCOOH) were added and the extract was washedwith the same extraction medium through a G 4 glass frit (JenaerGlass, Schott, D-6500, Mainz). The homogenate was dried at17°C in a rotary flash evaporator. The extracts containing theamino acids were dissolved in 1 x 1 ml and 2 x 0.5 ml lithiumcitrate buffer (pH 2.2) and used for ammonia and amino aciddetermination after removal of insolubles by centrifugation (1).For '5N determination, the effluent of the amino acid analyzer(Beckman Multichrom 4255, Beckman, D-8000 Muenchen) wascollected and analyzed as described by Berger and Fock (2).Enzyme Assays. Plant material used for these experiments was

grown in the greenhouse of the Botanic Gardens (University ofKaiserslautern) and adapted to phytochamber conditions for atleast 2 d. The gas exchange pattern of these plants indicatedCAM.The plant material was frozen in liquid N2 and ground with

sand and 20% (w/w) insoluble polyvinylpolypyrrolidone(Sigma). Enzymes were extracted at 0°C from Cissus stem sec-tions as described by McNally et al. (15) for leaves of twoKalanchoe' species except that PEG 20,000 was omitted. Thehomogenate was centrifuged for 10 min at 20,000g.

All assays were performed at 30°C for 30 min. Reactions wereterminated by placing the reaction tubes in boiling water for 1min. Identical samples heated at zero time were used as controls.The synthetase activity of GS was measured as described byRhodes et al. (20). GOGAT was assayed by determination ofglutamate formed during the MV-, NADH-, or NADPH-de-pendent reactions (14). The reaction mixture (total volume 1 ml)was identical with that of Matoh and Takahashi ( 14) except thatit contained elevated concentrations of 2-oxoglutarate (20 mM),MV (0.8 mM), or NAD(P)H (1 mM). One unit of enzyme activitywas defined as the amount of enzyme catalyzing the formationof 1 Amol glutamate/h. NAD(P)H-dependent GDH activitieswere measured in the assay medium of Nauen and Hartmann(18). The total volume was 1 ml and the activity of GDH wascalculated from the amount of glutamate formed (14).

Sources of Chemicals. '5NH4Cl and amido-'5N glutamine werepurchased from Amersham Buchler, D-3300 Braunschweig.MSO was obtained from Calbiochem Behring Co., La Jolla, CA92037; AZA was from Serva Feinbiochemica GmbH, D-6900Heidelberg. Other chemicals were obtained from E. Merck, D-6100 Darmstadt or Boehringer Mannheim GmbH, D-6800Mannheim.

RESULTSActivities of the key enzymes of ammonia assimilation, e.g.

GS, GOGAT, and GDH were present in extracts of stems of

Cissus quadrangularis (Table I). In vitro, the MV-dependentGOGAT activity and the GS activity hardly changed betweennight (phase I) and day (phase III). In contrast, the activities ofGDH and NADH-dependent GOGAT appeared to fluctuate.We were not able to detect NADPH-dependentGOGAT activity.The kinetics of net CO2 gas exchange into detached succulent

stem sections of C. quadrangularis are shown in Figure 1. Withrespect to pattern and rate the gas exchange of these internodesresembled that of otherCAM plants (10). These results were notquite compatible with the gas exchange data given by Ting et al.(23) who observed an unusual continuous CO2 uptake into stemsof this species also during the light-dark transition presumablydue to discontinous measurement. As a control in incubationexperiments, water was filled into holes punched through thefaintly green central pith of the stem. Although this treatmentaccounted for a tissue loss of approximately 10% of the sample,CO2 uptake declined by about 60% (Fig. 2, controls). As thechloroplasts are concentrated in the outer layers of the inter-nodes, we cannot explain the extreme loss of photosyntheticactivity at present. However, a substantical decrease in CAMactivity was also observed in wounded leaves ofKalanchoe (27).

Injection of 150 mm NH4C1 equivalent to an increase in theammonia pool by about 8 ,umol/g fw inhibited the successiveCO2 uptake and stimulated the CO2 evolution during the earlydark phase in accordance with the suggested acceleration of

Table I. Activities ofGS, GOGAT, and GDH in Extracts ofStems ofC. quadrangularis

In Vitro Enzymic Activity

Phase I Phase III

GS

GOGATMV

NADH

NADPHGDHNADH

NADPH

a Not detectable.

smol/gfw. h, ± SE2.10±0.07 1.57±0.14(n=2) (n=4)

2.56 ± 0.39 2.78 ± 1.60(n = 2) (n = 4)

0.75 ±0.10 NDa(n = 2)ND ND

3.75 ± 0.33(n = 2)

2.36 ± 0.14(n = 2)

1.70 ± 0.20(n=2)

0.63 ± 0.09(n = 2)

9 12 E 1TIME OFDAY (h)

FIG. 1. Diurnal pattern of CO2 gas exchange of detached stem sec-tions of C. quadrangularis at 200 Omol/m2 s 1 d after watering ( ), 2d after watering (- - -), and 3 d after watering ( ...). Means out of 14continuously measured replicates are shown. The maxima (± s) were3.13 ± 0.27, 2.56 ± 0.49, and 2.09 ± 0.47 jAmol/g fw during the first,second, and third dark period, respectively.

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Plant Physiol. Vol. 81, 1986

TIME OF DAY (h)FIG. 2. Effects of water, NH4C1, and MSO on the rates of net CO2

gas exchange of stem sections of C. quadrangularis at 200 M4mol/m2-s.Water (-), 150 mM NHCI (-- -), or 150 mm NHCI plus 5 mM MSO(.. .) were injected (50 lA/g fw) into holes punched through the centralpith of detached intemodes. Parental plants were watered 2 d before themeasurements. Arrows indicate the commencement of the incubationperiods. One of two very similar gas exchange experiments is shown.Injections during phase I or III had similar effects on the gas exchange.

0z 20F0i0

%20

PHASEI

P.2 o o

[4ASElE.

.laS.15ZPH4ASEEI

0 360 90120150

PERIOD OF NCUT0N (min)FIG. 3. '"N enrichments of glutamine (0, 0) and glutamate (, 0)

extracted from stem sections of C. quadrangularis after injection of '"Nlabeled (96 atom % "N), 150 mm "INH4CI (-, *), or 150 mM `NH4Clplus 5 mm MSO (0, E). 50 ,d/g fw solution were injected into a cut holein the longitudal axis of the stem.

glycolytic and respiratory processes during ammonium supply(8). Compared to incubations with NH4Cl, an injection ofNH4Clplus MSO (inhibitor of GS; 12) hardly influenced the gas ex-change of Cissus stems during the first 2 to 3 h of treatment, butfinally restricted CO2 uptake within 12 h completely (Fig. 2). Atpresent, we cannot decide whether this decline in net CO2assimilation is due either to accumulation of NH3 or to sideeffects ofMSO (1-3).The "N enrichments ofglutamine and glutamine, the primary

products of the alternative ammonia fixation pathways (GS/GOGAT or GDH), were analyzed after injection of 150 mM"5NH4Cl (Fig. 3). The "5N labeling pattern indicates that gluta-mine is the primary product of ammonia assimilation in thedark and in the light. As MSO suppressed the '"N labeling ofthese Cs amino acids almost completely, GS appears to play themajor role in the fixation ofammonia. Virtually the same resultswere obtained when these experiments were performed with 15

mm (instead of 150 mM) 15NH4C1.The 'sN labeling patterns are influenced by the pool sizes of

the amino acids and ammonia which are shown in Table II. Inaccordance with earlier findings (17), the amino acid concentra-tions did not change significantly in relation to the day periodthe samples were taken. However, the pool of ammonia fluc-tuated and was largest in the dark. MSO application increasedthe ammonium levels in various leaf tissues (2, 3, 9). In stems ofC. quadrangularis, the size of the total ammonia pool remainedalmost constant during MSO injections (data not shown). Thissuggests that MSO reached only the central region of the stems.During all incubations also the amounts of the amino acidsanalyzed hardly changed.The N transfer from glutamine, the product of GS activity,

into glutamate occurs via GOGAT activity in leaves of C3 andC4 plants (3, 9, 14, 29). For unknown reasons, injection of 15mM amido-"N glutamine suppressed net CO2 assimilation of C.quadrangularis after more than 2 h (Fig. 4). A similar gasexchange pattern was observed during incubations with gluta-mine plus AZA (inhibitor of GOGAT; 12). In contrast to theinhibition by glutamine Which was abolished after about 1 d, theAZA effect was not relieved during 3 d (data not shown).

Table III shows the '"N labeling of glutamate in internodesfrom C. quadrangularis treated with amido-"N glutamine in theabsence or presence of AZA. The low glutamine concentrationthat was injected did not substantially increase the total pool ofglutamine in the tissue, but made the analysis of the '5N enrich-ments of the amino acids rather difficult because of low 15Nlabeling. As GDH activity is favored by high NH3 concentra-

Table II. Average Concentrations ofAmino Acids and Ammonia in theStems ofC. quadrangularis during Three Metabolic Phases

Concentration ofAmino Acids during Phase

I III IV

,4mol/gfwAmmonia 3.13 ± 0.96a 1.63 ± 0.37 1.16 ± 0.29Serine 1.94 ± 0.32 1.65 ± 0.17 1.68 ± 0.41Asparagine 1.85 ± 0.50 1.48 ± 0.66 1.27 ± 0.25Glutamate 0.60 ± 0.21 0.53 ± 0.17 0.51 ± 0.19Glutamine 4.55 ± 1.52 4.24 ± 1.04 4.07 ± 2.02Glycine 0.46 ± 0.23 0.51 ± 0.18 0.69 ± 0.32Alanine 5.50± 1.12 4.45 ± 1.26 4.58 ± 1.82

' Standard deviation. n = 8 to 10 independent samples.

4-

o3

L 1

z

18 21 3 6 9 12 15 18TIME OFDAY (h)

FIG. 4. Effects of glutamine and AZA on the net CO2 gas exchangeof stem sections of C. quadrangularis. Water (-), 15 mm glutamine(- - -), or 15 mM glutamine plus 5 mM AZA (... .) were injected (50 ,1/g fw) into a cut hole in the longitudal axis of the stem. Parental plantswere watered 2 d before the measurements. Arrows indicate the com-mencement of the incubation periods. A typical run out of three repli-cates is shown. Injections during phase I or III had similar effects on thegas exchange.

PHASEIPHASE&I1 I PHASEI

I!~~~~~~~~~ldl!It'- ,.......

358 BERGER ET AL.

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AMMONIA FIXATION IN CISSUS QUADRANGULARIS

Table III. '5N Enrichments ofGlutamate in the Stems ofC.quadrangularis after Injections ofAmido-'5N Glutamine (96 atom %

'5N) or Amido-'5N Glutamine (96 atom % '5N) plus AZA

Glutamate '5N Enrichment duringTime Phase

I III IV

min atom % '5NA. Incubation with 15 mM amido-'5N glutamine (50 zd/g fw)

20 0.14 a 0.4140 0.35 0.19 0.3160 0.59 0.57 0.2390 0.69 0.78 1.05135 0.99 1.83 1.34

B. Incubation with 15 mM amido-'5N glutamine plus 5 mM AZA20 0.00 0.24 0.1140 0.32 a 0.3260 0.15 0.23 0.2190 0.13 0.05 a135 0.19 0.15 0.11

a Sample lost.

tions (16), fluxes of '5N via hydrolysis of the amido groupfollowed by GDH catalyzed NH3 fixation are expected to be lowin stems treated with low amino acid concentrations.'N was transferred from glutamine into glutamate, during the

metabolic phases I, III, and IV. AZA inhibited this flux almostcompletely during 135 min of injection (Table III).

DISCUSSION

This study demonstrates that ammonia assimilation proceedsvia the GS/GOGAT system in succulent stems of the Vitaceae,Cissus quadrangularis. Enzyme assays (Table I) confirmed pre-vious reports on the occurrence of significant levels ofGS (5, 15)and GOGAT (5) in CAM succulents. GDH occurs in the pho-tosynthetically active tissue of CAM plants (Table I; 5) and inthe stems of herbaceous plants (21), but it does not function asthe primary pathway for the assimilation of NH3 in the inter-nodes of C. quadrangularis. This conclusion is drawn from thefollowing observations: (a) although the pool size of glutaminewhich contains two nitrogen atoms was almost one order ofmagnitude larger than that of glutamate (Table II), the '5Nenrichments of glutamate were substantially lower than those ofglutamine in experiments with '5NH4C1 (Fig. 3). (b) MSO, usedin concentrations which do not affect GDH (12), suppressed thelabeling of glutamine and glutamate from '5NH4Cl (Fig. 3). (c)GOGAT was present in the tissue (Table I). Its involvement inthe conversion ofglutamine into the more universal amino groupdonor glutamate was indicated by the '5N transfer between theseamino acids as well as by the inhibition of that reaction by AZA(Table III).The labeling of glutamine and glutamate with '5N derived

from NH4Cl or amido-N of glutamine (Fig. 3; Table III) suggeststhat the GS/GOGAT system is active during all phases examined.In the light, '5N transfer between glutamine and glutamate hasbeen observed in spinach leaf discs (28, 29), wheat leaves (1, 3),and in other plants (16). 14C transfer between glutamine andglutamate was observed in wheat leaves (1, 25). In leaves ofDatura stramonium (13) and Zea mays (2) glutamine is themajor product of nitrogen fixation in the light.

In the central pith of internodes of C. quadrangularis GS wasstrongly inhibited by MSO (Fig. 3). In tissues of other plantsammonia accumulated in the presence of this inhibitor (1-3, 9,25), thus improving the conditions for optimum activity ofGDH(16). As the labeling of glutamate extracted from MSO treated

stems of C. quadrangularis was sometimes higher than that ofglutamine, GDH may have also been active under these condi-tions (Fig. 3). Although the "5N enrichments of ammonia in thetissue reached almost similar abundances in the light and in thedark, the '5N incorporated in the dark (about 0.3 umol g fw-'h-') was merely about half of that assimilated into glutamineduring illumination (about 0.8 gmol g fw-' h-'). In the dark,high '5N incorporation into glutamate in isolated spinach leafcells (7) or in spinach leaf discs (28) incubated in '5NH4Cl or 'INglycine, respectively, was ascribed to the deamination of gluta-mine. These authors argued that amido-N released from gluta-mine could be assimilated into glutamine via GDH.

Miflin and Lea (16) suggested that due to favorable Mg2"concentrations, energy charge, pH and reducing power availabil-ity, the GS/GOGAT system should be fully operative in thelight, but not in the dark. Knight and Weissman (1 1) found arhythmic activity pattern of GS in sunflower roots with maximain the light and in the dark. In chloroplasts isolated from etiolatedpea leaves, Matoh and Takahashi (14) observed that glutamatewas synthesized from glutamine both in the light and in the dark.Almost no glutamate formation occurred in darkened chloro-plasts from green leaves. Thus, these authors suggested that NH3assimilation in etiolated pea leaves might not proceed via Fd-dependent GOGAT commonly found in green leaves but byNADH-dependent GOGAT (14). On the other hand, Fd couldbe reduced by a NAD(P)H utilizing oxidoreductase and permitFd-dependent GOGAT to function in the dark (22). If the MV-dependent GOGAT activity measured in vitro (Table I) is relatedto the Fd-dependent activity of this enzyme in vivo, glutamatecould be formed from glutamine via NADH- or Fd-dependentGOGAT in Cissus stems during the dark period. From the datapresented here it is apparent that GS and GOGAT are operativein succulent tissue of the CAM plant C. quadrangularis both inthe light and in the dark.

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