enzymic assimilation of ii. reduction of nitrite to …enzymic assimilation of nitrate in tomato...

Enzymic Assimilation of Nitrate in Tomato PlantsII. Reduction of Nitrite to Ammonia'

G. W. Sanderson 2 and E. C. CockingDepartment of Botany, University of Nottingham, University Park, Nottingham, Great Britain

It is generally assumed at present that nitrate isreduced to the level of amino-nitrogen or ammonia,via nitrite (20, 21, 32). Support for this assumptioncomes from the demonstration of a widespread occur-rence of nitrate reductase in higher plants (27).However, as was pointed out previously (27), a finaldecision as to the validity of this assumption awaitsthe characterization of an enzyme system capable ofreducing nitrite to ammonia.

Nason, Abraham, and Averbach (22) in a briefreport stated that nitrite was reduced to ammonia, inthe presence of either DPNH or TPNH, and Mn++,by a partially purified soybean leaf extract. However,the results showed that significant quantities of am-monia were formed even in the absence of added ni-trite in spite of the fact that the enzyme preparationshould have been free of nitrate and nitrite due to thepurification carried out. Vaidyanathan and Street(29) reported cell-free enzyme extracts of excisedtomato roots which caused a disappearance of nitritein the presence of added DPNH and Mn+ +, but only2 % of the nitrite lost could be recovered as ammonia.Roussos and Nason (23) reported a highly purifiedenzyme from soybean leaves which would oxidizeDPNH and TPNH in the presence of nitrite or hy-droxylamine. However, nitrite did not disappear andthe product of hydroxylamine disappearance couldnot be identified. Huzisige and Satoh (13) have re-ported an enzyme from spinach leaves which willcause a disappearance of nitrite in the presence ofilluminated chloroplasts without the addition of anelectron carrier. The product of the reaction was notidentified. The reduction of nitrite was inhibited bythe addition of TPN. Hageman, Cresswell, andHewitt (10) were able to demonstrate that ammoniawas the product of nitrite reduction by a marrow leafenzyme preparation, but their system was dependenton the use of reduced benzyl viologen as an electrondonor. Neither DPNH nor TPNH could serve aselectron donor for this enzymic reduction in the ab-sence of benzyl viologen.

The present investigation was undertaken for thepurpose of clarifying these conflicting reports in orderto establish the role of nitrate reductase in nitrateassimilation in higher plants. During the course ofthe investigation additional information on the nature

1 Received Aug. 23, 1963.2 Present address: Tea Research Institute, Talawakele,

Cevlon.

of the enzymic system for the reduction of nitrite toammonia was obtained. For the purpose of this in-vestigation, identification of the product of nitrite re-duction was considered to be essential for the estab-lishment of the fact that nitrite reduction had oc-curred. A preliminary report of this work has beengiven (26).

Materials and Methods

Growth of Plant Material. Tomato plants (Lyco-persicon esculentunt, Mill., variety Sutton's Best-of-All) were used throughout this investigation. Plantculture was as described previously (27).

Enzymne and Grana Preparationi. Enzyme prepa-rations were made using the procedures describedpreviously (27). Gel filtration of enzyme prepara-tions was carried out as a routine during this investi-gation.

Grana were prepared according to a modificationof the method of Arnon, Allen, and Whatley (2). Allsteps after sampling were carried out in a cold roomat QO to 40 using cold apparatus and reagents. Theextraction media consisted of 0.32 -\ NaCl containingTris * HCl buffer (pH 7.5, 0.05 i) and 1 X 10-3 Mcysteine. Leaflets were removed from the rachis andground in a mortar with an equal weight of extractionmedia and about one-half their weight of acid washedsand. The macerate was strained through 4 layers ofcheesecloth and the filtrate was centrifuged for 1minute at 200 X g. The precipitate was discardedand the supernatant fraction was diluted 5-fold with1 X 10-3 M cysteine for use as the crude grana-enzyme preparation, G..

Washed grana preparations were made by centri-fuging the above undiluted supernatant for 10 minutesat 1750 x g. The supernatant fraction diluted 2.5-fold with 1 X 10-3 M cysteine containing the enzymeand a few grana was called E0 (crude enyme prepa-ration). The grana residue was taken up in theoriginal volume of extraction media and centrifugedagain for 10 minutes at 1750 X g. Finally, thisgrana residue was taken up in the original volume ofextraction media. The above 2 preparations made byresuspending the grana residues became preparationsG1 and G2, respectively, on 2.5-fold dilution with 1 X10-3 M cysteine.

Nitrite Reductase Assay I. Benzyl Viologen As-say. This assay was as described by Cresswell (6)and Hageman, Cresswell, and Hewitt (10).

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PLANT PHYSIOLOGY

Nitrite Redn ctase Assay II. Light-grana-enzymeAssav. This assay is a mlodification of the assay de-scribedl by Huzisige andl Satoh (13). The reactionmixture consiste(d of 1.0 nil grana-einzyme preparation,0.8 ml of Tris- HCl buffer (pH 7.5, 0.1 M). and 0.2ml of 2 X 10-3't NaNO2. Incubations w-ere car-ried out either in open test tubes or in evacuatedThunberg tubes placed in a water batlh at 28°. Thecomplete(d reaction mixtures were gently shakenperiodically to enisure uniform reaction conditions.Reaction mixtures Nwere illuminate(d with a 200-wOsram Sieray lamp held 25 cml above the water bath.The light intensitv on the reaction mixtures was about1300 ft-c. Incubations in the dark were carried outin tubes covered w\ith aluminum foil. Reactions werestoppe(l by placing reaction tubes in a briskly boilingwater bath for 3 miinutes. The tubes were then cooledin runniing tap wvater after which 0.2 ml of saturateduran-vl acetate and 2.8 nil of distille(d water wereaddlecl. The treated reaction mixtures wvere mlixe(d andtheni centifuged at 1000 X g for 5 nminutes. A suit-able aliquot of the superniatalnt, usuall- 1.0 mfl, wastaken for nitrite estimationi.

Nitrite Estimiiationi. Nitrite was estimate(d by a(dd-ing 1.0 ml of 1 (xv v ) sulfanilaimiide in N HCl and1.0 mll of 0.01 , (x/v) N-(1-naphthyl) ethylene-dliamiinie (lihvdrochloridle to a test solution containing2 to 70 niumoles of nitrite. The solutions were di-lute(d to 4.0 ml an(d mlixe(l. After 30 minutes theabsorptance at 540 ilgu xvas determined with a UnicamSP.500 spectrophotometer. The nitrite content was(letermineil from1 a stan(lar(d curve.

Ammonioa Estimiationi. Ammllonia wx as estimiaite(dafter separation froml reaction nmixtures by microdlif-fusion in Conwvav units (5). One nml of test solutionwvas place(l in the outer well of a Conwav unit con-taining 1.0 ml of 0.1 N HCI in the center xwell. Oneml of saturated borate-hvdroxide buffer (pH 10.1)(1) wvas intro(luce(d inlto the outer wxell to cause thedlistillatioln of ammiiioniia. Distillationi was allowe(d toprocee(l for just 4 hours. A set of standards was runwith each hatch. Finally, ammoniac was estimated onan 0.8 ml aliquot of the center xwell contents using thealkaline phenate-hyp-ochlorite metlho(d of Russell (24).

Wheni DPNH -\vas present in reactionimixtures itwxas necessary to miiake a special correction to am-mlonia estimlationis for the DPN which resulte(d fromDPNH oxidlationi. Both DPNH and DPN wverepairtially (legra(led with the release of ammliloniia un(lerthe alkaline conditions of the mlicro(liffusion. but DPNis significantly mlore labile than DPNH (cf. 31, 4, 18).The results of (letermillinig the anmmionia releasedl bye(lual amounts of D1'N and DPNH (luring miicro-diffusion with 3 (lifferent alkalis are shown in table I.It is clear froml these results that blank values foranimionia basedl on1 zero time conditions, i.e. when thecoenzyme is 100 %c reduced, are invalid as soon asDPNH oxidlation has commenced. Correction vasmade separately for each experiment by determliningthe ammlllonia blank for reaction mixtur-es \with thecoenzyme totally re(luce(d (iinitial conlditions withDNPH- 0 '< oxidized ) and(I with the coenzvmie to-

tally oxidized (DPNH - 100 % oxidized). Thesepoints wvere used to plot a correction chart by drawinga straight line between them. The percent DPNHoxidized was determined for each reaction mixture atthe tinme of its sampling for ammonia estimation bymeans of its absorptance at 340 m,u (12). With thisquantity the appropriate blank could be read off thecorrection chart.

Nitrate Reduictase Assav. This enzyme was as-sayed as described previously (27).

Hill Reaction Actizvity Assay. Hill reaction activ-ity wvas determined by the method of Jagendorf (15).

Protein1 Estimilation1. Protein was estimated bythe method of Lowry et al. (19).

Results

Nitrite Redutctase Activity tsinig Redulced Bezl3t1Viologeni. as Electron, Donor. It wvas found earlierthat nitrite was not lost fronm cell-free extracts oftomilato plaints w,hen either DPNH or TPNH wereuse(d as the electron donor during a stu(ly of nitratere(luctase (27). Before proceeding with an investi-gation of the in vivo nitrite redlucing system, it was(lesirable to demonstrate that the extracts under in-vestigation (lid in fact contain an active enzyme systemcapable of carrying out this process. The reducedbenzvl viologen assay describedI by Hageman, Cress-xvell, and Hewitt (10) was used for this purpose.Using this assay, an active nitrite reducing system w\asreadily shox-vn to exist in these extracts. Typical re-sults are shown for a leaf extract in figure la and fora root extract in figure lb. Both extracts were Sepha-dex treatecl to remove endogenous nitrate so as toeliminate nitrate reductase activity which would alsoprocee(l in the presence of reduced benzyl viologen.

The pro(luction of ammonia (luring the above as-says paralleled nitrite loss stoichiometrically as shownin figures la andl lb. There vas nIo loss of nitrite orproduction of ammonia un(ler the conditions of theassay in the absence of the enzyme preparation or inthe presence of enzymle preparations wvhich had firstbeen heate(d to 1000 for 3 minutes.

Nitrite Reductase Activiitv Usinig DPNH andTPNH as Electron Donor. During a stu(ly of nitratere(luctase, no loss of nitrite was found un(ler the con-ditions of the nitrate reductase assay use(l with eitherDPNH or TPNH as electron donor (27). How-ever, in view of published reports vhich state thatnitrite reductase is an enzyme requiring reduced pyri-(line nucleotidles (22, 23, 29), a concerted effort wasmladle to findl an assaY sy-steml int which enzymic nitritere(luction wvould occur in the presence of these re-(luced coenzymes.

The standard nitrate reductase assay describedpreviously (27) was modified by replacing the 10,umole KNO, with 0.1 ,mole NaNO2 for use as thebasic assay systenm, and the effect of added cofactorswas tested by adding one-tenth ,umole of the follow-ing cofactors per assay: FAD. FMN, MnCL2, Vita-nin K3, recluced glutatlhionie. anid sodiunm ascorbate(pH 7.5). The cofactors Nvere teste(d wvith both

424

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SANDERSON AND COCKING-NITRITE TO AMMONIA

t700

ED600

0

400

14>1

1v300

01 0

z

Z 200

0

E

>2

O

.

*~~~~-

-~~~~~~~~~/o

/ o~~~

0/

/0

oo~~~~~~~~~~~~~~~~.

a

I I I I~~~~~~~~0 10 20 30

TIME (min)

FIG. la. The relationship between nitrite reduction andammonia accumulation in the essay of nitrite reductase ina tomato leaf preparation with reduced benzyl viologen.The enzyme was extracted from leaflets of 66-day oldplants and purified by Sephadex treatment. Each ml ofenzyme preparation contained 3.65 mg protein. 0, Nitritereduced and O, ammonia accumulated.

DPNH and TPNH as electron donor but no nitriteloss occurred in any case. The following buffers werealso substituted in the standard assay for the sodiumphosphate buffer (pH 7.5, 0.1 M) normally used:

Table IThe Effect of 3 Different Alkalis on the Yield

of Aimmionia from DPN and DPNH during 6-HourAIMicrodiffusion in Conway Units

Form of Pyridine Yield of NH3*Nucleotide AlaiRtgN~N

unit Conway unit)

DPN 0.5 N NaOH 8.42DPN Saturated Solu- 2.00

tion ofK2C03

DPN Borate-hydroxide 0.63buffer pH 10.1

DPNH 0.5 N NaOH 1.67DPNH Saturated Solu- 0.30

tion ofK2C03

DPNH Borate-hydroxide 0.08buffer pH 10.1

* These are the average of duplicate determinations.

O 13 30

TIME (min.)

FIG. lb. The relationship between nitrite reduction andammonia accumulation in the assay of nitrite reductase ina tomato root enzyme preparation with reduced benzylviologen. The enzyme was extracted from roots of 70-dayold tomato plants and purified by Sephadex treatment.Each ml of enzyme preparation contained 1.75 mg protein.0, Nitrite reduced and 1, ammonia accumulated.

Sodium phosphate buffer (pH 7.0, 7.5, and 8.0, 0.1 MI),sodium pyrophosphate buffer (pH 7.0, 7.5, and 8.0,0.1 m), and Tris HCl buffer (pH 7.5, 0.1 Nf). Nonitrite loss occurred in any case.

The level of DPNH was varied between 0.91 X10-3 M (the level in the standard assay) to 2.73 X10-3 i. As the level of DNPH was raised therewas a significant loss of nitrite but this was shownto be entirely nonenzymatic. Catalytic amounts ofoxidized benzyl viologen (0.05 ,umole/ml of reactionmixture) were added to standard nitrite reductaseassay reaction mixtures containing either DNPH or

TPNH. The assays were carried out anaerobicallyin evacuated Thunberg tubes. Nitrite was lost enzy-mically when either DPNH or TPNH were used as

electron donors (table II).Nitrite Reduction by a Grana-enzyme System.

The investigation thus far had shown that the apo-

enzyme(s) involved in nitrite reduction to ammoniawere present in an active state in extracts of tomatoplants but that neither DPNH nor TPNH could serve

as the electron donor under a variety of assay condi-tions without the addition of benzyl viologen. Atten-tion was next directed to assay systems containingparticulates in the hope that a method of producingthe reducing agent required for nitrite reduction couldbe found.

Huzisige and Satoh (13) have reported nitritedisappearance in an assay system containing granaand a soluble enzyme. Since these workers did notreport the pxoduct of nitrite reduction it is not certain

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PLANT PHYSIOLOGY

Table IIEffect of Catalytic Amnou0nts of Benzyl V'iologent onNitrite Reduction in a Reaction Mixtuire Containiing

a Tomnato Leaf Enzkymiie PreparationLeaflets of 60-day old tomato plants were extracted

and purified by Sephadex treatment. The enzyme prep-aration contained 3.73 mg protein/ml. Assays were car-ried out under anaerobic conditions for 30 minutes at 28°using the following reaction mixture: 1.5 ml 0.1 M sodiumphosphate buffer pH 7.5, 0.3 ml. 2 X 10-3 sodium ni-trite, 0.15 ml. 1 X 10-3 M benzyl viologen (or equalvolume of distilled water), 0.3 ml 5.4 X 10-3M DPNHor TPNH (or equal volume of distilled water), 0.6 mlenzyme preparation and distilled water to bring totalvolume to 3.0 ml.

Benzyl vio- Nitrite re- AmmoniaBoenuzymlevia duced mu~ accumulated*mlogeactmone uoedinzymeay moles/ml my moles/mlml reaction used in assay enzyme prep- enzyme prep-mixture aration aration

0.05 DPNH 115 -350.05 DPNH 143 -130.05 NONE 0 00.05 TPNH 258 1920.05 TPNH 184 1480.05 NONE 0 0None DPNH 0 0None TPNH 0 0

*, Negative value indicates loss of ammonia.

100 I a

e/%

bC

CLa.U 80C

EN/

' 60 /

(I /0 0.4,

40 .3oE

I-

> 0

.(0

__

< O

TIME (min.)

FIG. 2. The effect of light on nitrite reduction by an

enzyme-grana preparation from tomato leaves. 0, Illu-minated and Z1, dark assays. No additions of eitherDPNH or TPNH were made to these assays.

that they actually studied a nitrite reductase as theyclaimed. However, these results appeared to beworthy of further investigation.

Crude grana-enzyme preparations from tomatoleaves were assayed in the light anid in the dark. Theresults of a typical experiment are shown in figure 2.In the light there was a rapid initial loss of nitritewhich took place in less than 60 seconds followed bya more gradual loss. No nitrite loss occurred inilluminated tubes containing a grana-enzyme prepara-tion which had been heated at 100° for 3 minutes priorto the addition of nitrite. In the (lark there was a lossof nitrite equal to that found in the light after 60seconds but no additional reduction of nitrite tookplace.

These results indicated that the natural electrondonor, CoX, was present in the grana-enzyme prep-arations in a partially or wholly re(luced state. Theenzymic reduction of nitrite was extremely rapid untilthe supply of CoXH2 was use(d up. This wouldaccount for the initial rapi(d reduction of nitrite foundl.Furthermore, it may be h3ypothesized that the granacontain the mechanism required to reduce CoX whenthey are illuminated.

In a separate series of experiments it was foundthat ammonia accumulation accompanied nitrite loss(fig 3). The presence of succinate in assay reaction

10 20TIME (min. )

FIG. 3. The relationship between nitrite loss anid am-monia accumulation during nitrite reductase assay of anenzyme-grana preparation from immature tomato leaves.Preparations were made from the leaves of 58-day oldplants and 30, moles of succinate solution (adjusted topH 7.5 with sodium hydroxide) were added to each assay.0, NH3 accumulated in the light (anaerobic incubation);upper 0, NO2- lost in the light (anaerobic incubation);0, NH3, accumulated in the light (aerobic incubation);*, NO2- lost in the light (aerobic incubation) lower 0,NH3 accumulated in the dark (aerobic incubation); g,NO- lost in the dark (aerobic incubation),

426




mixtures was required for ammonia accumulation insome cases but not in others. The effect of succinateon ammonia accumulation was not studied in detailbut it appeared to have a function in regulating con-version of endogenous carbohydrates to a-ketogluta-rate through the regulation of the citric acid cycle inthe presence of limited amounts of DPN (25). Asituation which appears to be analogous has been de-scribed by Jones and Gutfreund (16). The enzymewhich could account for a disappearance of ammonia,L-glutamate dehydrogenase, has been shown to bepresent throughout the tomato plant by VanDie (30).

Grana-enzyme preparations were made using 4levels of cysteine in the extraction media. The re-

sults of assays of the 4 preparations which were

carried out according to the standard procedure are

shown in figure 4. A cysteine concentration of 1 X

*; 1000

S-

t,

E 4

la

0

<

L.

l60

0

> 2C

o0 x 10-4 M

LEVEL OF CYSTEINE IN THE* EXTRACT-IONMEDIUM

FIG. 4. The effect of cysteine level in the extractionmedium on nitrite reductase activity in enzyme-granapreparations from tomato leaves.

10-3 M was decidedly the most favorable for the ex-

traction of an active nitrite reductase system.The results of a comparison of aerobic and anaero-

bic conditions on nitrite reductase activity are shownin figure 3. Under anaerobic conditions the activitywas greater than under aerobic conditions and theeffect was accentuated as the length of the incubationwas increased.

A comparison was made of sodium phosphate andTris HCl buffers (pH 7.5, 0.1 M) as reaction mix-ture buffers in the standard assay. There was no

difference in the enzymic activity due to the use ofeither of these buffers.

The effect of substrate concentration was studied

c

.2 8o

AL

40.

0

.E

60

0 200 400 600 ;8; lOOO

NITRIT-E CONCENTRATION(mJJ moles / mi. reacti on mixture)

FIG. 5. The effect of substrate concentration on nitritereductase activity in an enzyme-grana preparation.

in standard light-grana-enzyme assays modified byvarying the nitrite concentration. and shortening theincubation period to 5 minutes (fig 5). The resultsshow that nitrite above the level of 2.5 X 10-4 Mwas inhibitory to nitrite reductase. This inhibitionof nitrite reductase by nitrite at concentrations aboveabout 250 m,jumoles per milliliter is a rather unusualfeature of the system. The Michaelis constant fornitrite was determined graphically (17) and it wasfound to be 2.7 X 10-6 M.

The effect of grana-enzyme concentration wasstudied using the standard assay modified by varyingthe amount of grana-enzyme preparation used. Inevery case, the reaction mixture was made up to afinal volume of 2.0 ml by the addition of distilled wateras required. Under these conditions the activity in-creased exponentially with grana-enzyme concentra-tion up to 0.8 ml of preparation per assay (fig 6).The nearly exponential increase in enzyme activityobtained with increasing enzyme-grana concentrationis probably due to an osmotic effect on the integrityof grana brought about by the osmotic gradient exist-ing in the assays because of the use of distilled waterto adjust reaction mixture volumes.A typical set of results obtained from the assay

of a grana-enzyme preparation, its fractions, and re-constituted systems are shown in figure 7. A highdegree of light stimulated nitrite reduction was lostduring fractionation (compare assay of G. with assayof EO, G1, and G.,). It was not possible in these trialsto recover the activity on reconstitution (compareassay of G. with assay of E. + G, or Eo + G,).The latter result was obtained even though G, andG, were found to contain high Hill reaction activity

Lindicating the preservation of certain functional in-

Hi [VA-

427

L4



PLANT PHYSIOLOGY

> 100~0/a

@ 80

L/

ON60/z

E

20-20

o 02 0.4 06 0.8 1.O

ENZYME-GRANA CONCENTRATION(mi./assay )

FIG. 6. The effect of enzyme-grana concentration on

nitrite reductase activity.

tegrity, and even though it was known that E0 con-

tained nitrite reductase which was active with re-

duced benzyl viologen.The results shown in figure 7 are seen to be due to

the effect of the fractionation treatments on the integ-rity of the grana. The increase in CoXH2 in the re-

constituted systems, E0 + G1 and E0 + G2, is

60

60

-

e' 40

z

e 20E

IE t0

>70 10 20 0 10 20

Go Eo

1l1n I 10I0 10 20010 20

TIME (min.)

G, G2PREPARATION ASSAYED

0 10 20 0 10 20

Eo. GI Eo t G,

FIG. 7. Nitrite reductase activity in an enzyme-granapreparation, in its fractions, and in reconstituted systems.

G. (crude enzyme-grana preparation), E. (crude enzyme

preparation), G1 (crude grana preparation), G2 (washedgrana preparation), Eo + G1 (crude enzyme preparationplus crude grana preparation) and Eo + G2 (crudeenzyme preparation plus washed grana preparation).

shown by the high level of nitrite reduction in theassays carried out for zero time or in the dark. Thisindicates a loss of structural integrity with a con-

comitant release of CoXH2 hich had been enldog-enous to the grana. Further, the inability of thesesystems to bring about an increase in the amount ofnitrite reduced with time suggests that the ability ofthe grana to reduce CoX in the light has been lost.

The Distributiont of Nitrate Assimilatory Activityin the Tomlato Plant. Five-month-old tomato plantswere sampled for their roots, immature fruits, petiolesand leaf rachii, leaflets of immature leaves and leafletsof mature leaves. Cell-free preparations were madefrom these samiples using the method described underMaterials and Methods for the preparation of grana-

enzyme suspensions in order to preserve the particu-lates of the tissues. The 5 preparations obtained were

then assayed for nitrate and nitrite reductase activi-ties. All nitrite reductase assays were as for illumi-nated enzyme-grana systems (table III). No addi-tions of coenzyme were made to either nitrate or ni-trite reductase assays.

Nitrate reductase was found to be present in all of

the 5 preparations. The rates of activity found mustbe taken as minimal ones because of the presence ofsome nitrite reductase activity.

A rapid initial loss of nitrite occurred in all prepa-

rations as shown by the zero minute (i.e. placed in a

boiling water bath as soon as possible after additionof the enzyme-grana preparation assays). However,this loss then decreased during the next 15 minute in-cubation in the assays of all preparations except thosefrom leaves. No nitrite loss occurred in tubes con-

taining enzyme preparations which had been heatedat 1000 for 3 minutes prior to the addition of nitrite.This rapid initial loss of nitrite in all cases showedthat the reducing agent, CoXH2, and the enzyme,nitrite reductase, were present in all 5 preparations.However, only in the case of the leaf preparations was

a mechanism for replenishing the supply of CoXH2operative. In all other preparations, after the initialloss of nitrite, nitrate reductase activity was sufficientto replace the nitrite lost. Succinate was addled toone set of assays in an attempt to promote the reduc-tion of nitrite. The result, however, was to promotenitrate reductase to the extent that any nitrite reduc-tase activity was masked. This is shown by the factthat with all 5 preparations there was less nitrite lostin the presence of succinate than in its absence.

Discussion

Extensive trials to obtain an enzyme system fromthe tomato plant capable of reducing nitrite using re-

duced pyridine nucleotides as electron donors were

unsuccessful. This was so in spite of the presence ofan enzyme capable of reducing nitrite to ammonia asshown by assays with reduced benzyl viologen,Hageman, Cresswell, and Hewitt (10) have reportedsimilar results using marrow leaf extracts.

Earlier reports of enzymic nitrite reduction de-pendent on reduced pyridine nucleotides (22, 23, 29)

428

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I I,)L-.LA-J- -



SANDERSON .AND COCKING-NITRATE TO AMMONIA

Table IIIDistribution of Nitrate and Nitrite Reductase Activity in a Mature (5-Month old) Tomato Plant

All extracts were made using 0.32 M Sodium chloride containing 0.05 M Tris * HCI buffer pH 7.5 and 10-3 M cysteine,so as to maintain the integrity of grana and other cellular particulates as far as possible.

Nitrate reductase Nitrite reductase Nitrite reductase Nitrite reductase activity*activity activity activity (15 min assay)

(15 min assay) (Zero time assay) (15 miii assay) with 0.2 ml of M succi-Source of enzyme m,amoles NO2- mAmoles NO2- mumoles NO2- nate pH 7.5 added to

accumulated/mg reduced/mg reduced/mg each assay. mu/moles NO2-protein protein protein reduced/mg protein

Roots 10.3 7 0 -26Fruit 3.6 7 2 - 9Petioles and leaf rachii 2.4 11 10 -12Immature leaflets 3.6 12 44 33Mature leaflets 10.8 14 25 20

* Negative values indicate nitrite accumulation.

appear to be best explained in other ways. The am-monia production reported by Nason, Abraham, andAverbach (22) was likely to have been due to am-monia released in the degradation of DPN during themicrodiffusion phase of their ammonia estimationprocedure. The stimulatory effect of manganese ionsand FAD on ammonia production was most likely dueto their stimulatory effect on pyridine nucleotide oxi-dase activity (14, 8, 9). This would increase theamount of DPNH oxidation and the concomitant in-crease in alkali labile DPN would cause an apparentrise in the amount of ammonia in the reaction mix-tures. The level of pyridine nucleotide oxidase activ-ity was found to be very high in tomato plant extracts(25) and in marrow plant extracts (6), and in allprobability it is present in soybean extracts as well.

Vaidyanathan and Street (29) reported briefly ona nitrite reductase using DPNH as electron donorwhich required manganese ions. However, onlyabout 2 % of the nitrite lost was recovered as am-monia.

The pyridine nucleotide-nitrite enzyme describedby Roussos and Nason (23) was not capable of re-ducing nitrite and hence-it is of questionable signifi-cance in nitrite assimilation. It has been pointed outby Hageman, Cresswell, and Hewitt (10) that theseresults appear to be best explained in terms of aperoxidase system such as has been described byCresswell and Hewitt (7).The results of adding catalytic amounts of oxidized

benzyl viologen to nitrite reductase assay mixtureshowed that nitrite was lost from these assay systemswhen either DPNH or TPNH were used as electrondonors (table II). TPNH supported the greatestamount of activity. However, only when TPNH wasused as electron donor did ammonia accumulate.

Hageman, Cresswell, and Hewitt (10) found asimilar situation in extracts of marrow leaves exceptthat there was nitrite reduction only when TPNHwas used as the electron donor. These workers sug-gested that a TPNH dependent diaphorase (15) wasacting to transfer electrons from TPNH to benzylviologen and that the latter substance acted as a co-factor in the reduction of nitrite. This appears to

explain the results obtained in this investigation withthe following amplification: A) The diaphorase intomato is active with either DPNH or TPNH. Suchan enzyme has been reported to be present in tobaccoroots (28). B) The benzyl viologen appears to beacting in a linked enzymic reduction chain and not asa true cofactor. C) A DPNH-specific L-glutamatedehydrogenase (3) was active in these preparations(30) and this prevented the accumulation of ammoniain the presence of DPNH. (c.f. 29).

The results obtained using an illuminated enzyme-grana system suggest that either the actual coenzymeinvolved in nitrite reduction or an electron carriercapable of reducing nitrite in the presence of nitritereductase and its associated coenzyme is present ingrana. This substance remains unidentified and ithas been called CoX (reduced form CoXH2). Evi-dence to show that CoX does not reside exclusivelyin grana but that it is present in cellular particulatesof all organs comes from the results of assays of ex-tracts of 5 different organs of tomato plants whichshow that there is a rapid initial loss of nitrite in thepresence of enzymes and particulates (table III).

The results obtained in this investigation withnitrite reduction in light-grana-enzyme systems havesuggested the following set of reactions to explain themechanism of nitrite reduction in vivo:

nitritereductase

3CoXH2 + NO.- + H1+ >(dark)

NH3 + 3CoX + 2H.,O (I)grana

CoX + H.,O > CoXH2 + >202. (II)(light)

Reaction II is analogous to the Hill reaction whereCoX is an unidentified naturally occurring Hill re-agent. Furthermore, there is a possibility that CoXrepresents more than one substance.

These results agree with and extend the earlierfindings of Huzisige and Satoh (13) who reportednitrite reduction in the presence of a soluble enzymeand a grana preparation, both obtained from spinach

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PLANT PHYSIOLOGY

leaves. These workers found a low rate of nitrite re-duction in the dark which they attributed to a de-hydrogenase activity which acted in place of illumi-nated grana to reduce the electron carrier. However,in view of the fact that their preparations were par-tially purified and therefore virtually free of sub-stances which could act as substrates for a dehydro-genase, and in the light of the present findings, it ap-pears more likely that this reduction in the dark wasdue to a gradual breakdown of the grana in the hypo-tonic assay reaction mixtures with the concomitantrelease of the reduced electron carrier, CoXH2, whichhadl been endogenous to the grana.

The identity of the natural electron carrier in-volved in nitrite reduction remains somewhat uncer-taini although it appears that ferredoxin may be theelectron carrier for the grana system (11). How-ever, the results indicate that it is a substance presentin all organs of the tomato plant and not present onlyin the grana system. That nitrite reductase activitycan be found wherever nitrate reductase is found(table III) supports the contention that nitrate re-ductase is indeed involved in nitrate assimilation.The rates of nitrite reductase activity in light-grana-enzyme systems found so far are considerably lowerthan those of nitrate reductase (the activity of theformer was about one fifth to one eighth the activityof the latter). Since nitrite is only rarely detectablein plants, it must be assumed that the conditions ofthis assay are suboptimal for nitrite reductase.

Summary

A cell-free nitrite reductase system obtained fromtonmato plants was capable of bringing about the rapid(within 60 seconds) reduction of nitrite in the dark,but illuminated grana were necessary for sustainedactivity. The presence of 1 X 10-3 M cysteine in thegrana-enzyme preparation was essential for maximumactivity. The Michaelis constant for nitrite was2.7 X 10-6 M and ammonia was the product of nitritereduction.

The enzyme would reduce nitrite in the absenceof grana when reduced benzyl viologen was used asthe electron donor. Reduced pyridine nucleotideswere ineffective in bringing about nitrite reduction.The natural electron donor is unidentified but it wasshown to be present in all organs of the tomato planttested.

The demonstration of the presence of a naturalenzyme system capable of reducing nitrite to ammoniain plant organs verifies the supposition that nitratereductase is involved in nitrate assimilation in higherplants.

Acknowledgments

The Fulbright Scholarship held by one of us (G.W.S.)during the course of this investigation is gratefullyacknowledged. A Special Research Grant from D.S.I.R.to E. C. Cocking, and a gift of TPNH from the SigmaChemical Co., St. Louis, are also gratefully acknowledged.This work formed part of a thesis approved for the degreeof Ph.D. in the University of Nottingham.

Literature Cited1. ARCHIBALD, R. M. 1943. Quantitative microdeter-

mination of ammonia in the presence of glutamineanid other labile substances. J. Biol. Chem. 151:141-48.

2. ARNON, D. I., M. B. ALLEN, AND F. R. WHATLEY.1956. Photosynthesis by isolated chloroplasts. IV.General concept and comparison of 3 photochemicalreactions. Biochim. Biophys. Acta 20: 449-61.

3. BULEN, W. A. 1956. The isolation and characteri-zation of glutamic dehydrogenase from corn leaves.Arch. Biochem. Biophys. 62: 173-83.

4. COLOWICK, S. P., N. 0. KAPLAN, AND M. M. Ciorri.1951. The reaction of pyridine nucleotide withcyanide and its analytical use. J. Biol. Chem.191: 447-59.

5. CONWAY, E. J. 1957. Microdiffusion Analysis andVolumetric Error. 465 p. Crosby-Lockwood,London.

6. CRESSWELL, C. F. 1961. An investigation into ni-trate, nitrite, and hydroxylamine metabolism inhigher plants. 186 p. Ph.D. Thesis, University ofBristol, England.

7. CRESSWELL, C. F. AND E. J. HEWITT. 1960. Oxida-tion of hydroxylamine by plant enzyme systems.Biochem. Biophys. Res. Commun. 3: 544-48.

8. HACKETT, D. P. 1956. Pathways of oxidation incell-free potato fractions. Plant Physiol. 31:111-18.

9. HACKErT, D. P. 1958. Pathways of oxidation incell-free potato fractions. II. Properties of thesoluble pyridine nucleotide oxidase system. PlantPhysiol. 33: 8-13.

10. HAGEMAN, R. H., C. F. CRESSWELL, AND E. J. HE-WITT. 1962. Reduction of nitrate, nitrite, and hy-droxylamine to ammonia by enzymes from higherplants. Nature 193: 247-50.

11. HEwriT, E. J. AND G. F. BETTS. 1963. The re-duction of nitrite and hydroxylamine by ferre-doxin and chloroplast grana from C(icurbita pepo.Biochem. J. 89: 20 P.

12. HORECKER, B. L. AND A. KORNBERG. 1948. Theextinction coefficient of the reduced band of pyridinenucleotides. J. Biol. Chem. 175: 385-90.

13. HUZISIGE, H. AND K. SATOH. 1961. Photosynthe-tic nitrite reductase. I. Partial purification of theenzyme from spinach leaves. Botan. Mag. (Tokyo)74: 178-85.

14. HUMPHREYS, T. E. AND E. E. CONN. 1956. Theoxidation of reduced diphosphopyridine nucleotideby Lupine mitochondria. Arch. Biochem. Biophys.60: 226-43.

1i. JAGENDORF, A. T. 1956. Oxidation and reductionof pyridine nucleotides by purified chloroplasts.Arch. Biochem. Biophys. 62: 141-50.

16. JONES, E. A. AND H. GUTFREUND. 1962. Glutamatesynthesis and the control of reactions linked withthe nicotinamide-adenine dinucleotide coenzyme inmitochondria. Biochem. J. 84: 46-51.

17. LINEWEAVER, H. AND D. BURK. 1934. The deter-mination of enzyme dissociation constants. J. Am.Chem. Soc. 56: 658-66.

18. LOWRY, 0. H., J. V. PASSONEAU, AND M. K. ROCK.1961. The stability of pyridine nucleotides. J.Biol. Chem. 236: 2756-57.

19. LoWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, ANDN. J. RANDALL. 1951. Protein measurement withthe Folin phenol reagent. J. Biol. Chem. 193:265--75.

430




20. NASON, A. 1956. Enzymatic steps in the assimila-tion of nitrate and nitrite in fungi and higher plants.In: Inorganic Nitrogen Metabolism, p 109-36.Johns Hopkins Press, Baltimore.

21. NASON, A. 1962. Symposium on metabolism oforganic compounds. II. Enzymatic pathways ofnitrate, nitrite, and hydroxylamine metabolism.Bacteriol. Rev. 26: 16-41.

22. NASON, A., R. G. ABRAHAM, AND B. C. AVERBACH.1954. The enzymatic reduction of nitrite to am-monia by reduced pyridine nucleotides. Biochim.Biophys. Acta 15: 159-161.

23. Roussos, G. G. AND A. NASON. 1960. Pyridinenucleotide-nitrite and -hydroxylamine enzymesfrom soybean leaves. J. Biol. Chem. 235: 2997-07.

24. RUSSELL, J. A. 1944. The colorimetric estimationof small amounts of ammonia by phenol-hypochlor-ite reaction. J. Biol. Chem. 156: 457-61.

25. SANDERSON, G. W. 1962. The enzymic assimilatiornof nitrate in the tomato plant. 186 p. Ph.D. Thesis,University of Nottingham, England.

26. SANDERSON, G. W. AND E. C. COCKING. 1963. Theenzymic assimilation of nitrate in the tomato plant.Biochem, J. 87: 28 P.

27. SANDERSON, G. W. AND E. C. COCKING. 1964. Theenzymic assimilation of nitrate in the tomato plant.I. Nitrate reductase. Plant Physiol. 39: 416-22.

28. SISLER, E. C. AND H. J. EVANS. 1958. TobaccoSci. 2: 132-38.

29. VAIDYANATHAN, C. S. AND H. E. STREET. 1959.Nitrate reduction by aqueous extracts of excisedroots. Nature 184: 531-33.

30. VAN DIE, J. 1962. The distribution of glutamicdehydrogenase activity and a-ketoglutarate invarious parts of the tomato plant. Acta. Botan.Neerl. 11: 1-10.

31. VON EULER, H., F. SCHLENK, H. HEIWINKEL, ANDB. HOGBERG. 1938. Z. Physiol. Chem. 256:208-28.

32. WEBSTER, G. C. 1959. Nitrogen Metabolism inPlants. Row, Peterson Biol. Mono., Evanston,Illinois. 152 p.

The Glyoxylate Cycle in Maize Scutellum 1.2Ann Oaks and Harry Beevers

Department of Biological Sciences, Purdue University, Lafayette, Indiana

In contrast to fat-storing seeds, cereals have aready supply of carbohydrate, which after an initiallag is transferred to the developing embryo (10).Nevertheless there is a progressive loss of lipid ma-terial from cereal scutella during germination (7,13).If it is assumed that fatty acids are released fromstorage lipids, and that they are further degraded byfl-oxidation to acetyl CoA, then the addition of ace-tate-C'4 should trace the final stages of fatty acidoxidation. The results of the present investigationshow that part of the acetate carbon is incorporatedinto scutellar and embryo sugars, when acetate isapplied locally at the scutellum. In addition highlevels of isocitrate lyase (isocitritase) and malatesynthetase were found in extracts prepared frommaize scutella.

MethodsSeeds of Zea mays (hybrid variety Wf9 x 38-11)

were soaked in water for 12 hours, and allowed togerminate for an additional 48 hours on moist absorb-

'Received Aug. 28, 1963.2This work was supported by Contract AT-11-1330

with the United States Atomic Energy Commission.

ent paper at 30'. At this stage the endosperm wasremoved and the scutella of the detached embryoswere placed in a salts solution (14) containing 1 %glucose and acetate-2-C'4 (about 0.1 ,umole/embryo;2.5 X 107 cpm; sp. act. 7.3 mc/mM) for 2 hours.The embryos were then washed and placed in a freshglucose-salts solution for an additional 10 hours.After each time interval (2 hr and 12 hr) samples of10 scutella or 10 roots were extracted with 80 %(v/v) ethanol. The ethanol extract was dried underreduced pressure at 40° and the lipids were extractedwith ethyl ether. The remaining residue was takenup in water and passed through Dowex-50 (H+)and Dowex-1 (formate) resins to separate basic,acidic, and neutral components. Samples from eachfraction were dried on nickel-plated planchets and theC14 content determined with a thin-window continuousgas-flow Geiger-Miiller tube. Samples of the alco-hol insoluble residue and ether soluble material werecombusted to CO, (16), and collected as BaCO3,which was plated on microporous planchets forcounting.

The sugars in the neutral fraction were separatedby 1-dimensional paper chromatography in 1-butanol:

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enzymic assimilation of ii. reduction of nitrite to …enzymic assimilation of nitrate in tomato...

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