THEACTIVITY OFVegetal, Serrano, 113, Madrid, Spain. 3 Present address: New Yorlk State College of Agri-culture, Agricultural Experiment Station, Cornell Uni-versity, Ithaca, New York

.MOLYBDENUMI AS A PLANT NUTRIENT. X. SOMNE FACTORS AFFECTING\THE ACTIVITY OF NITRATE REDUCTASE IN CAULIFLOWER

PLANTS GROWN WITH DIFFERENT NITROGENSOURCES AND WMOLYBDENUM LEVELS

IN SAND CULTURE1

M. I. CANDELA,2 E. G. FI$HER3 AND E. J. HEWITTAGRICULTURAL RESEARCH COUNCIL UNIT OF PLANT NUTRITION (MICRONUTRIENTS),

LONG ASHTON RESEARCH STATION, UNIVERSITY OF BRISTOL, EXNGLA-ND

Nitrate red(ietase enzymes were obtained in par-tially purified states from Neurospora crassa an(l soy-bean by Nason and Evalns (16) and Evans and Nason(8) who showed also that the enzyme is widely dis-tributed with greatlv differing activity in severalhigher plants. The presence andI role of molybdenumas a specific component of these enzymes was shownby Nicholas and Nason (17, 18, 19)

Evidence has been presented elsewhere by Agar-wala (1), Agarwala andl Hewitt (3), Hewitt and 'Mc-Cready (13) and Nicholas, Nason and McElroy (19)that molybdenum is also required by plants and fungiwhen provided with nitrite, ammonium compounds,urea, or other sources of nitrogen and that the roleof molybdenum in nitrate re(luctase may not en-tirely explain its function aIs an essential element.This conclusion, together with the evidence of Ev-ansand Nason (8), who reported that they obtained noresponses to different nitrogen sources or molybdenumsupplies in regard to their effects on the activitv ofnitrate reductase, suggested that further investiga-tion of some of the factors affecting the activity ofthe enzyme was desirable.

The investigations reported here were made oncauliflower, as this plant has been extensively usedat Long Ashton in previous studies concerning mo-lybdenum, with special reference to the origin of thedisorder knowN-n as whiptail (2, 3, 10, 11).

MIATERIALS AND iIETHODSCauliflower plants (Brassica oleacea L. var. botry-

tis cv. Majestic) wAere grown in sand culture usingmethods alreadcy fully described (3, 9). Nitrogen wasgiven as nitrate or as nitrite or ammonium sulphateusually at 12 millimoles/liter. Molybdenum wasgiven at 0.05 ppm, 10 to 20 ppm or was omitted. Theresidual level in the molybdenum omitted treatmentswas probably the equivalent of 0.00001 to 0.00002ppm. Plants grown at this level developed typicalsevere symptoms of whiptail already described (2, 3).The symptoms were prodluced in all three nitrogentreatments but plants grown with nitrate showedalso the characteristic mottling and necrosis thatoccur under these conditions (3, 13).

1 Received December 18, 1956.2 Present address: Instituto de Edafologia y Fisiologia

Vegetal, Serrano, 113, Madrid, Spain.3 Present address: New Yorlk State College of Agri-

culture, Agricultural Experiment Station, Cornell Uni-versity, Ithaca, New York.

EN ZYMIE EXTRACTION AND SA-MPLING: 'Nitrate re-

ductase was extracte(l by grinding fresh tissue for15 minuites with neutral acid-washed silica sandl andlthree times its weight of extractant at 0) C in achilledl mortar. The extracting reagent in 1954 was0.1 MI phosphate buffer pH 7.0 containing 10A4 1\cysteine and ethylenediamine tetraacetate (EDTA)(18). In 1955 the buffer pH was 8.8 The groundtissuies were filtered through muuslin and the extractwras centrifuged at 20,000 x g at 0° C and kept in iceor used immediately. Activity observed in these ex-tracts was also compared with that obtained by grind-ing at 00 C in mechanically driven close-fitting glassmacerators, by disintegration in a high speed blendor,or by maceratingf in a hand operated Ten-Broeck pat-tern g1lass macerator.

In 1954 the leaf lamina of entire plants was useedexcept that the two oldest or other senescent leavesandl those under about 2 cm in length were discarde(l.In 1955 plants were subdivided into pairs of leavesfrom adjacent nodes and into root, stemii andl petiolewhich were examined separately. It as, not possibleto saimple at one time from all treatments. Simul-taneous comparisons were made in regard to molyb-denum status whilst the nitrogen treatments weresampled consecutivelY on three or more occasions inran(lom order. In some tests the plant materi.l wasstored at -18° C before assay but the same storagetreatment was used in all comparative tests. Theredi(d not appear to be any- loss of nitrate reductaseover 24 hours under these conditions.

NITRATE REDUCTASE ASSAY AND PREPARATION OF

REAGENTS: Two tenths or 0.5 ml of extract w-ere use(lalmost immediately in the following reaction system:1.0 ml 5 % sodium nitrate; 0.1 ml reduced (liphospho-pvridine nucleotide (DPNH 1 mg/ml); 0.05 ml boiledlextract of pig heart acetone powder; 2.5 or 2.8 ml of0.2 M phosphate buffer pH 7.3; distilled water to pro-duce 5.15 ml. The assays were carried out at 270 Cfor 20 minutes in 1954 or for 10 minutes at 280 C in1955. The respective rates of reaction were constantover these intervals. Nitrite was estimated as (le-scribed bv Evans and 'Nason (8) with the additionof sufficient hydrochloric acid to obtain maximumcolour development. Blank estimations were carriedIout by omitting DPNH and all assays were made induplicate. Activities were expressed as total activity:millimicromoles of nitrite produced in 10 or 20 min-utes per 0.2 or 0.5 ml of extract and as specific ac-tivitv: millimicromoles nitrite in one minute per mg

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CANDELA ET AL-MOLYBDENUM AS A PLANT NUTRIENT

TABLE IEFFECT OF METHOD OF GRINDING ON NITRATE REDUCTASE

ACTIVITY IN CAULIFLO-WER LEAF EXTRACTS *

RATIO OF BUFFER TO LEAF WTEXPT METHOD OFGRINDING 1/3 1/4 1/5

1 MoIrtar 154 ... 200N.I.R.D. blendor ... ... 177

2 Mortar 133 ... ...

N.I.R.D. blendor ... ... 140MIechanical glass 91 47 ...

3 MIortar 84 ... 110Mechanical glass 26 37 20

4 Mlor tar' 96 97 102Ten-Broeck 59 ... 80

5 Mortar 137 ...Mechlanical glass 73 ... ...

6 Moirtar 179 ...

Mechanical glass alone 133 ... 136W'ith ground glass 79 ... 58

* Millimicromoles nitrite produiced in 10 min per gmleaf wt.

protein. Protein was estimated colorimetricallv bythe biuret reaction (21) or turbidimetrically in 1954,and turbidimetrically after precipitation with 2 % tri-chloracetic acid in 0.06 M ammonium sulphate in1955.

The presence of nitrite in solutions of analyticalreagent grade sodium nitrate and other reagents wasminimised by preparing daily a fresh solution of ni-trate to which 0.5 mg ammonium chloride was addedfollowed by boiling for three minutes, and also byselecting reagents for minimum nitrite content. TheDPNH was prepared from 60 to 75 %. DPN ob-tained from y-east or commercially. It was reducedin 0.04 M pyrophosphate buffer containing 0.2 Iethyl alcohol by addition of crystalline alcohol de-hydrogenase prepared from yeast as described byRacker (20). The pH was maintained between 9.8and 10.0 with 2N KOH during the reaction whichwas observed spectrophotometrically at 340 m/A. Ap-proximately 100 mg actual DPN were used in 70 mland about 70 to 80 % of the DPN was reduced. Atthe conclusion of the reaction the solution was heatedfor 5 minutes in a boiling water bath, cooled rapidlyand stored at -18° C. The pig heart extract used toprovide FAD (8) was prepared by boiling one partof an acetone powder of pig heart in three parts ofwater for 5 minutes, centrifuging and storing at- 180 C.

ACID PHOSPHATASE ASSAY: Acid phosphatase ac-tivity was also determined in some extracts. Thetwo enzymes showed markedly different trends in ac-tivity in regard to the regions from which theyw-ere extracted. This disposed of the possibility thatlarge differences observed between the activity ofnitrate reductase in samples from different regionsl11ight be dutie merely to a variable degree of disrup-

tion of the cells in different extractions. Acid phos-phatase was assayed as the rate of hydrolysis ofphenolphthalein phosphate at 270 C over a 15-minuteperiod at pH 5.4 in 0.075 M acetate buffer. The re-action w-as stopped bv adding 1 ml 15.5 % trichlor-acetic acid. The colour of liberated phenolphthaleinwas developed on addition of 1 ml N sodium hy-droxide to bring the solution to pH 10 approximatelyand was measured with a Spekker absorptiometerwithin 10 minutes, using green filters and a calibra-tion curve prepared from phenolphthalein.

RESULTSEFFECT OF GRINDING PROCEDURE AND PH: Results

in table I show that grinding in a mortar with sandor in the high-speed blendor gave the highest ac-

2 3 4 5 6 7 8 9 O I1 12 13 14 15STEM NODE

FIG. 1. Nitrate reductase in different regions of cauli-flower plants grown with nitrate.

281

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PLANT PHYSIOLOGY

FIGa2

4 5 6 7 8 9 10 11 12 13 14 IS

STEM NODE

I-

-i-I0

I--

iA-

U)

LL

CLJ

16

FIG. 2. Effects of leaf position and molybdenum supply on distribution of nitrate reductase activity andprotein in cauliflower grown with nitrate. Mean curves for 3 separate experiments shown as squares, circles andtriangles, respectively.

FIG. 3. Effects of leaf position and molybdenum supply on distribution of nitrate reductase activity andprotein in cauliflower grown with nitrite. Mean curves for 3 separate experiments shown as squares, circles andtriangles, respectively.

tivities. Extracts prepared with the glass macera-

tors were, however, usually low in activity and theinclusion of ground glass appeared to be still more

unfavourable. The ratio of extracting fluid to tissueweight over a range between 3/1 and 6/1 did notgreatly affect the activity per unit leaf weight.

The activity of the centrifuged extract was greatestwith a sharp optimum between pH values of 7 and7.4 in phosphate buffer for cauliflower grown withnitrate in contrast with purified soya bean enzymefor which the optimum was about 6 (8).

DISTRIBUTION OF ACTIVITY AND PROTEIN: The ac-

tivities observed were the net activities without cor-

rection for effects of nitrite reductase. Preliminarytests indicated that nitrite reductase activity was rel-atively high in roots and low in aerial parts under the

conditions of assay. Inclusion of hydroxylamine hy-drochloride from one half, up to double the amountused by Evans and Nason (8) to inhibit nitrite re-

ductase did not appreciably affect the observed ac-

tivity of nitrate reductase in leaf extracts. It wasconcluded that nitrite reductase activity of these ex-

tracts was not sufficient to interfere with the generalconclusions reached in this work.

In plants given nitrate with molybdenum (figs 1,2), the net activity was greatest in mature fully ex-

panded, though not senescent leaves, adjacent to theoldest two or three leaves remaining on the plant atthe time. Net activity decreased progressively inyounger leaves. The distribution in leaves of plantsgrown with nitrite or ammonium sulphate and mo-

lybdenum (figs 3, 4) was similar to that in plants

282

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CANDELA ET AL-MOLYBDENUM AS A PLANT NUTRIENT

given nitrate, but the decrease from the maximumwas relatively less sharp in young leaves of plantsgiven ammonium sulphate. In plants aged 16 to 20weeks the maximum activity was usually found inleaves at nodes 6 and 7 or 8 and 9 (figs 1, 2, 3).

The distribution of net nitrate reductase activityin petioles (fig 1) resembled that in leaves with some-what lower values. In stems the maximum occurredin younger sections than in leaves. No nitrite pro-duction could be detected using extracts of roots.Measurements on loss of nitrite in the presence andabsence of added nitrate, suggested that nitrate re-ductase activity was low in roots.

The distribution of extractable protein differedfrom that of nitrate reductase and increased fromoldest to youngest leaves (fig 1 to 4). The changeswere marked in laminae and small in petioles. Theprotein extracted from stems was the same for lowerand mid-stem sections and increased markedly in theyoungest stem region. The distribution of specificactivity resembled that of total activity, but thechanges were relatively smaller between old regionsand those with maximal activity and relatively greaterbetween these and the young regions. Specific activ-ity figures were especially high for petioles owing tothe low protein values (fig 1).

AGE OF PLANT: The net total activities in leavesof plants grown with 10 to 20 ppm molybdenum andthe three sources of nitrogen in 1954 (table II) wereconsiderably less for plants aged 20 weeks than thoseaged 12 weeks sampled at the same time. The spe-cific activities, however, were similarly in both agegroups for nitrite and ammonium treatments due to

~--1

0u>-,LL' 0a: 1- 0

{LO3><

2 A

z

- I0

F I G.4

4 5 6 7 8 9 10 11 12 13 14 15STEM NODE

FIG. 4. Effects of leaf position and molybdenum sup-

ply on distribution of nitrate reductase activity andprotein in cauliflower grown with ammonium sulphate.Mean curves for 3 separate experiments shown as

squares, circles and triangles, respectively.

TABLE IIEFFECTS OF NITROGEN SOURCE AND AGE OF PLANT ON

NITRATE REDUCTASE ACTIVITY IN LEAVES OFCAULIFLOWER GROWN IN 1954

NITROGEN AGE OF TOTAL PROTEIN, SPECIFICSUPPLY PLANTS, ACTIVITY * MG/0.5 ACTIVITY **WKS ML

NitrateNormal level 12 21.6 3.2 1.68

20 12.5 2.3 1.36Double level 20 17.7 3.2 1.38

Nitrite 12 15.8 3.4 1.1620 13.5 2.8 1.20

Ammon. sulphate 12 10.4 3.0 0.8720 5.9 1.7 0.87

* Millimoles nitrite in 10 min per 0.2 ml extract.** Millimoles nitrite in 1 min per mg protein.

the lower protein extracted from the older plants,but specific activity also was decreased in the olderplants grown with nitrate.

EFFECTS OF NITROGEN SOURCE AND MIOLYBDENUMILEVEL: The net total and specific activities (figs 1to 4, tables II and IV) were decreased in the ordernitrate > nitrite > ammonium sulphate often withsignificance at 0.1 % in plants aged 12 weeks given10 ppm molybdenum in 1954. In tests with plantsaged 20 weeks the total activities were similar withnitrate and nitrite and much less with ammoniumsulphate. Plants aged 20 weeks given double thenormal nitrate level had higher total and similar spe-cific activities compared with those given the lowerlevel. In 1955 the maximum values and distributionof net total activities were similar in plants givennitrate or nitrite in the presence of 0.05 ppm molyb-denum. In plants given ammonium sulphate themaximum values were between one-third and one-sixth of those observed in the other two nitrogentreatments (figs 2, 3, 4). The changes in amounts ofprotein extracted from leaves at successive nodeswere similar in the three nitrogen treatments in thepresence of molybdenum and the effects of nitrogensource on relative specific activities resembled in gen-eral their effects on total activities.

In the absence of molybdenum (figs 2, 3, 4) thedifferences in net total activities between the differentnitrogen sources were smaller. Values in leaves ofplants given nitrite or ammonium sulphate, however,were perceptibly greater than those in plants givennitrate, possibly because the protein extracted fromsuccessive leaves was much less in the nitrate treat-ment than in the other two. For the same reasonthe specific activities were similar for the differentnitrogen treatments in the molybdenum deficiencyseries.

Omission of molybdenum markedly decreased nettotal nitrate reductase activity in nitrate and nitritetreatments (figs 2, 3), but the effect was much smallerin plants given ammonium sulphate, where valueseven with molybdenum, were generally low (fig 4).

NH4 +Mo -0 0 --MO

Cr. ."

C,

5

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283

rd



PLANT PHYSIOLOGY

Total activities were distributed more uniformly indeficient than in normal plants, and maxima wereless marked especially in plants given ammoniumsulphate. In plants given nitrate without molyb-denum (fig 2) activity usually increased from oldestto youngest leaves and net activities in old leaveswere sometimes apparently negative. This was at-tributed to a slight excess of nitrite reductase ac-tivitv over that of nitrate reductase in these plants.The relative effect of molybdenum status on nitratereductase activity varied greatly and ranged from aratio of over 15 to 1 to less than 1.5 to 1 in differ-ent regions of plants grown with or without molybde-num. The maximum values in molybdenum deficientplants tended to occur in leaves that were attached athigher nodes than those on normal plants.

Molybdenum deficiency markedly decreased theprotein extracted from leaves of plants grown withnitrate (fig 2). Smaller differences were also ob-served in plants grown with nitrite or ammonium sul-phate where values observed in leaves from any pairof nodes on molybdenum deficient plants often com-pared fairly closely with those in leaves from thepair of next older nodes on the normal plants (figs 3,4). Specific activities were also generally decreasedby molybdenum deficiency especially in plants grownwith nitrite or ammonium sulphate, and did not showthe marked maximum values at intermediate stemnodes observed in normal plants (figs 3, 4).

EFFECT OF LEAF INFILTRATION BY MOLYBDATE ONNITRATE REDUCTASE AND ACID PHOSPHATASE: Leaveswere removed from molybdenum deficient plants and(divided into half laminae. One half was infiltratedseveral times whilst immersed in a 5 x 10-; M solutionof sodium moly\bdate by alternate evacuation andadmission of air, until apparently watersoaked, andl

TABLE IIIEFFECTS OF INFILTRATION OF PAIRED HALF-LEAVES OF MO-LYBDENUM DEFICIENT CAULIFLOWER PLANTS WITH 105 M

MOLYBDATE ON NITRATE REDUCTASE ACTIVITY *

INFILTRATION TREATMENTSOURCE OF NITROGEN

LEAF TREAT- WATER MOLYBDATEMENTRANGE MEAN RANGE MEAN

Random Nitrate 1.0-10.8 3.85 - 1.4-21.4 6.60leaves

Random Nitrite 1.2- 7.7 4.04 5.3-14.1 9.08leaves

Leaves fromsuccessivenodes

(i) Nitrite 3.3- 7.2 5.14 2.6-12.8 7.28(ii) 2.7-10.1 5.68 2.7- 8.9 5.90(iii) " 3.7- 9.1 6.40 5.6-10.8 8.67

Random Ammon. 2.0- 7.2 4.60 3.0-11.4 8.22leaves sulphate

* Millimicromoles nitrite produced in 10 min by 0.5ml extract.

I -

G.CD z

ZW- 0.JO

' U,

-JzL o

I Iz°-iE i

FIG.5

5 6 7 8 9 10 11 12 13 14 15 16STEM NODE

FIG. 5. Effect of leaf position on distribution of acidphosphatase activ-ity in 8 sets of assays with cauliflowerplants grown with nitrite. Paired half leaves:A * * Infiltrated with molybdate.+ X AZ X Infiltrated with water.

the other half was similarly infiltrated with water as

a control. The fluid absorbed was determined bythe change in weight. Before extraction the leaveswere kept in a saturated space for 18 to 24 hours dur-ing which they remained turgid and eliminated thesurplus water. Several tests were made with singleleaves chosen at random from lower nodes, and a fewtests were mnade in which a series of leaves was sam-

ple(l in relation to stem nodes. The results are sum-marised in table III. Infiltration by moly bdenumincreased the activity in most of the tests regardlessof the nitrogen treatment but the magnitude of theresponse varied greatly in different leaves. No re-

sponse to molybdenum as observed when it was

added at the time of maceration. Mlarked increasesin nitrate reductase activitv have been observed inwhole plants and detached leaves two to three hoursafter introducing molybdenum or nitrate in later ex-

periments by E. J. Hewitt.Acid phosphatase activity was measured in the in-

filtration experiments and results are shown in figoure5. The distribution was quite distinct from that ofnitrate reductase and closely resembled that for pro-

tein shown in figures 2, 3, and 4. The acid phospha-

tase vallues increased in a regular manner from old toyoung leaves and suggested that the extraction pro-

cedure used wN-as valid and reproducible. No effect ofmolv bdate infiltration on acid phosphatase activitywas observed.

EFFECT OF LIGHT AND DARKNESS ON NITRATE RE-DUCTASE: These tests were prompted bv two consid-erations, namely (i) the possibility that prolonged(larkness might cause sufficient protein loss to de-creaIse enzyme activity; and (ii) the conclusion byBurstr6m (6) and others, that light has a specificfunction in nitraLte reduction in some plants. Plants

08 /

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06- ~ ~ NO

a A

oA A

0

C p pp-I I a

284

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CANDELA ET AL-MOLYBDENUMN AS A PLANT NUTRIEN-T

NITROGENAS

N03ON02 0NH4 X-IN LIGHTIN DARKNESS---

0 2 4 6DAY S

F 1IG.6

1-:I..1-u

O 2ecn

I0

wI.-202a. I

2 L

0 2 4 6DAYS

FIG. 6. Nitrate reductase activity in leaves of cauli-flower grown with different sources of nitrogen in day-light or darkness.

aged 13 weeks which hadl been grown with nitrate,nitrite or ammonium sulphate at the high molyb-denum levels in 1954 were placed in darkness in thegreenhouse or were otherwise allowed to grow in thenormal way. Total and specific nitrate reductase ac-tivities were determined in triplicate samples fromnormal and darkened plants at two-day intervals.The results are shown in figure 6. There were markeddecreases in the total and specific activities signifi-cant at the 0.1 % level for plants of each nitrogentreatment whilst they were kept in darkness. Totalextractable protein did not show changes of corre-spondcing magnitude. Similar results were observedwith plants aged 21 weeks in the second experiment(fig 7) which was not replicated because of shortageof material. It was found that when normal lightwas restored there were rapid increases in the totalaind specific activities in plants from each nitrogentreatment. The changes that were detectable duringthe first three hours of daylight continued over a

TABLE IVEFFECTS OF 48 HOURs EXCLUSION OF AIR ON NITRATE

REDUCTASE ACTIVITY IN- LEAVES OF CAULIFLOWERPLANTS AGED 13 WEEKS (1954 EXPT)

NITROGEN ATMOS- TOTAL PROTEIN, SPECIFICSUPPLY PHERE ACTIVITY * MG/0.5 ML ACTIVITY **

Nitrate Air 22.0 3.1 1.77N2 11.1 3.6 0.77

Nitrite Air 18.5 3.4 1.36N2 8.0 2.8 0.71

Ammon. Air 9.0 3.4 0.66sulphate N2 8.0 3.9 0.51

* Millimoles nitrite produced in 10 min by 0.2 mlextract.

** Millimoles nitrite produced in 1 min per mg pro-tein.

period of 24 hours until the following day but themain increases occurred in the first 12 hours. Therealso appeared to be a net synthesis of protein duringthe first 6 hours of daylight, which produced slightlyhigher values than those observed initially.

EFFECTS OF EXCLUSION OF AIR: As the mainte-nance of organised metabolic activity sometimes de-pends on normal respiration, the effects of exclusionand reintroduction of atmospheric oxygen were tested.Plants grown with each of the nitrogen treatmentswere enclosed in transparent airtight plastic coversthat were sealed around the outside of the pots. Ni-trogen was passed rapidly through a tube into theinterior to displace air and a slow stream of nitrogenwas then maintained to exclude air as far as possiblefor 2 days. At the end of this period the plants weresampled and nitrate reductase activity was estimated.Results for plants aged 13 weeks in December, 1954are given in table IV. In the second experiment inFebruary 1955 with plants aged 21 weeks, the changesthat occurred after the restoration of normal atmos-pheric conditions were also observed and are shownin figure 8. Total and specific net activity of nitratereductase was greatly decreased by exclusion of air.

0 DAYS

FIG. 7. Effect of darkness and restoration of liglht on

nitrate reductase activity in leaves of cauliflower grown

with different sources of nitrogen.

20

6

12

8

I.4

-i

0

4

0

285

5

1 A24



PLANT PHYSIOLOGY

0

50 N03 m

I N02 c)

40 NH4.*

30

20

IO'AIR0612

0 48HOUR S

FIG.8

z

0

FIG. 8. Effect of exclusion and re-admission of airon nitrate reductase activity in leaves of cauliflowergrown with different sources of nitrogen.

The activity was also rapidly restored on re-admit-ting air to the plants. No consistent trends wereobserved for the amounts of extractable protein.

DISCU-SSIONThe niet nitrate reductase activity in cauliflower

is determined by several factors. The pronouncedpeak in activity associated with mature but not senes-cent leaves of normal plants is noteworthy. This dis-tribution was practically independent of the type ofnitrogen nutrition and clearly distinct from that ofanother enzyme, acid phosphatase. The distributionof total extractable leaf protein and nitrate reductaseare relatively independent at some stages, althoughin ageing plants total activity fell as leaf protein de-creased. The total activities observed in cauliflowerappeared to exceed considerably those reported byEvans and Nason (8) for soya bean and many otherplants when the data are recalculated on a compar-able basis. Maximal activity occurred in the pri-mary and young trifoliate leaves of soya bean in con-

trast to the mature leaves of cauliflower.The effect of molybdenum supply was consistent

with existing knowledge regarding the role of molyb-denum in the enzyme (17, 18). The failure of Evansand Nason (8) to observe an effect of molybdenumon the enzyme in young soya bean plants might havebeen due to the molybdenum reserves in large seededlegumes which may delay deficiency effects for a wholegeneration (12). It is also apparent that the effectof molybdenum may be small or negligible in regionspossessing only minimal activity. It was found in

cauliflower, as in fungi (19) that the enzyme as suchis apparently not produced by tissues that have de-veloped in the absence of sufficient molybdenum andcannot be immediately reactivated in disrupted cells.Work with fungi (19) has shown that 3 hours are re-

quired before the synthesis of new enzyme can bedemonstrated after the addition of molybdenum to

deficient mycelia. The activity in cauliflower wasalso increased when detachedl leaves were infiltratedwith molybdate and increases have been observedsince by Hewitt after 2 hours. In intact tomatoplants the effect of adding molybdenum may be ob-served in two hours as nitrite production from theaccumulated nitrate in the deficient tissues (22). Asimilar response to the addition of molybdenum toexcised discs from molybdenum deficient cauliflowerplants may also be inferred from experiments on res-piration by Ducet and Hewitt (7). The effects ofgrowing the plants with different sources of nitrogenalso suggest adaptive features in the production ofthe enzyme. As with fungi (19), production of theenzyme was much greater in the presence of nitrateor nitrite than of ammonia, but was not completelysuppressed by this last treatment as observed in thefungi (19).

Evans and Nason (8) suggested that the effectof light on nitrate reduction described by Burstr6m(6) was consistent with the photochemical reductionof pyridine nucleotides by illuminated chloroplastsand their utilisation by nitrate reductase. The resultsobtained here show that exclusion of light from theplants may cause also a temporary loss of enzymeactivity in extracts even when adequate reduced pyri-dine nucleotides are present. In this connection Stov(23, 24) recently concluded that added riboflavincatalyses a photo-reduction of nitrate in irradiatedwheat leaf extracts and provides a more effective elec-tron donor for nitrate reductase in this system thanreduced pyridine nucleotides. There are other ex-amples also of the effects of light on enzyme produe-tion, as for example the observation of Applemanand Pyfrom (4) that red and blue light exert dif-ferent effects on catalase production by etiolatea bar-ley plants.

Analogous changes to those observed in darknessand light were observed when air was excluded andre-admitted, but it cannot yet be concluded that theywere in fact due to the same cause in both sets oftreatments. Since this work was ended, Morton (15)observed independently that anaerobic conditionscaused similar reversible changes in nitrate reductioncapacity in fungi. He concluded that the enzymesresponsible wvere not lost and resynthesised but wereonly reversibly masked by the anaerobic conditions.Removal of free carbohydrate from the medium oraddition of ammonia produced similar reversible ef-fects to those of anaerobic conditions. Lenhoff, Nich-olas and Kaplan (14) have found that the relativeproportions of flavin and haematin enzymes in Pseu-domonas fluorescens also are determined by atmos-pheric conditions as well as by the available metalsin the culture medium. Such changes contrast mark-edly with the tendency for enzymes such as inver-tase and peroxidase to persist even after extensiveproteolysis in detached leaves kept in darkness as ob-served by Axelrod and Jagendorf (5). It may beconcluded from this work that nitrate reductase is ahighly labile enzyme in living cells as well as in vitro.

286

A



CANDELA ET AL-MOLYBDENUM AS A PLANT INUTRIENT

Work is now in progress, using this enzyme as amodel, to investigate adaptive aspects of enzyme ac-tivity in higher plants as revealed by changes in nu-tritional or environmental conditions.

SUMMARY1. Cauliflower plants cv. 'Majestic were grown in

sand culture with deficiency, normal or excess levelsof molybdenum and with nitrogen supplied as ni-trate, nitrite or ammonium sulphate.

2. Nitrate reductase was extracted by grindingleaves with sand in a mortar or by maceration in ahigh speed blendor with buffer solution at 00 C.These methods gave comparable activities but grind-ing in mechanically driven close fitting glass macera-tors produced low activities. The optimum pH wasabout 7.2 in phosphate buffer extracts.

3. The net nitrate reductase activity was maximalin mature leaves and was markedly lower in bothsenescent and rapidly expanding young leaves.

4. Extracts from leaves possessed greater net totalactivity than those from stems or petioles, and thatof roots was extremely low. The specific activity permg protein was much greater in petioles than inleaves or stems.

5. The nitrogen source also determined the levelof nitrate reductase activity. This was high in plantsgiven nitrate and usually high in those given nitrite.Plants given ammonium sulphate possessed markedlylower but not negligible activities.

6. In regions showing maximal activity, plantsgrown without molybdenum possessed much lower nettotal and specific nitrate reductase activities than thenormal plants. In regions showing minimal activitythese differences were small. Plants given excess mo-lybdenum did not appear to possess more activitythan normal plants. The effect of molybdenum sup-ply mentioned above was greatest w-ith plants givennitrate and least with those given ammonium sul-phate.

7. Infiltration of molybdenum into leaves of de-ficient plants resulted in increases in nitrate reduc-tase activity in 18 to 24 hours.

8. Nitrate reductase activity was greatly depressedby prolonged darkness during two to six days andrecovered rapidly within a few hours of restoring theplants to light.

9. Exclusion of air for 48 hours also caused markeddecreases in activity; and readmission of air led torapid recovery in activity.

10. Changes in activity were not closely related tothe total soluble protein content of the tissues. Thedistribution of the enzyme in plants grown with dif-ferent molybdenum levels and nitrogen sources wasmarkedly different from the distribution of extract-able protein which was, however, similar to that ofanother enzyme-acid phosphatase.

The tenure of a Joint Overseas Studentship of theConsejo Superior de Investigaciones Scientificas MIa-drid and of the British Council, London, by M. I.

Candela in 1953-55 and the grant of Sabbatic leavein 1935 to E. G. Fisher which respectively enabledthem to participate in this work are gratefully ac-knowledged.

We wish to thank MIr. G. A. Clarke for the sta-tistical analy-sis of the data for 1954 and AMessrs. G.J. Dickes and AM. W. Heyes for technical assistance.

LITERATURE CITED1. AGA.RW^-ALA, S. C. Relation of nitrogen supply to the

molybdenum requirement of cauliflower grown insand culture. Nature 169: 1099. 1952.

2. AGARWALA, S. C. and HEwITT, E. J. Molybdenumas a plant nutrient. III. The interrelationships ofmolybdenum and nitrate supply in the growth andmolybdenum content of cauliflower plants grownin sand culture. Jour. Hort. Sci. 29: 278-290. 1954.

3. AGARWALA, S. C. and HEWITT, E. J. Molybdenumas a plant nutrient. VI. Effects of molybdenumsupply on the growth and composition of cauli-flower plants provided with different sources ofnitrogen in sand culture. Jour. Hort. Sci. 30: 163-180. 1955.

4. APPLEMAN, D. and PYFROM, H. T. Changes in cata-lase activity and other responses induced in plantsby red and blue light. Plant Physiol. 30: 543-549.1955.

5. AXELROD, B. and JAGENDORF, A. T. The fate of phos-phatase, invertase and peroxidase in autolysingleaves. Plant Physiol. 26: 406-410. 1951.

6. BURSTR6M, H. Photosynthesis and accumulation ofnitrate by wheat leaves. Annals Agr. Coll., Sweden11: 1-50. 1943.

7. DUCET, G. and HEWITT, E. J. Relation of molybde-num status and nitrogen supply to respiration incauliflower leaves. Nature 173: 1141-1142. 1954.

8. EVANS, H. and NASON, A. Pyridine nucleotide ni-trate reductase from extracts of higher plants.Plant Physiol. 28: 233-254. 1953.

9. HEWITT, E. J. The Use of Sand and Water Cul-tures in the Study of Plant Nutrition. Common-wealth Bureau of Horticulture, East Malling, Tech.Comm. No. 22. 1952.

10. HEWITT, E. J. and JONES, E. W. The production ofmolybdenum deficiency in plants in sand culturewith special reference to tomato and Brassicacrops. Jour. Hort. Sci. 23: 254-262. 1947.

11. HEWITT, E. J. and BoLLE-JONES, E. W. Molybde-num as a plant nutrient. I. The influence of mo-lybdenum on the growth of some Brassica crops insand culture. Jour. Hort. Sci. 27: 245-256. 1952.

12. HEWITT, E. J., BOLLE-JONES, E. W. and MILES, PEGGYThe production of copper, zinc and molybdenumdeficiencies in crop plants grown in sand culturewith special reference to some effects of watersupply and seed reserves. Plant and Soil 5: 205-222. 1954.

13. HEWITT, E. J. and MCCREADY, C. C. Relation ofnitrogen supply to the molybdenum requirementof tomato plants grown in sand culture. Nature174: 186-187. 1954.

14. LENHOFF, H. M., NICHOLAS, D. J. D. and KAPLAN,N. 0. Effects of oxygen, iron and molybdenumon routes of electron transfer in Pseudomonasfluorescens. Jour. Biol. Chem. 220: 983-995. 1956.

15. MORTON, A. G. A study of nitrate reduction infungi. Jour. Exptl. Bot. 7: 97-112. 1956.

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16. NASON, A. and EVANs, H. Tliphosphiopyiidine nu-cleotide-nitrate reductase in Neurospora. Jour.Biol. Chem. 202: 655-673. 1952.

17. NICHOLAS, D. J. D. and NASON, A. Molybdenumand nitrate reductase. II. Molybdenum as a con-stituent of nitrate reductase. Jour. Biol. Chem.207: 353-360. 1954.

18. NICHOLAS, D. J. D. and NASON. A. Role of molyb-denum as a constituent of nitrate reductase fromsoy bean leaves. Plant Physiol. 30: 135-138. 1955.

19. NICHOLAS, D. J. D., NASON, A. and McELROY, W. D.Molybdenum and nitrate redtuetase. I. Effect ofmolybdenum deficiency on the Neurospora en-zyme. Jour. Biol. Chem. 207: 341-351. 1954.

20. RACKER. E. Crystalline alcohol dehydrogenase from

b)aker's yeast. Jotur. Biol. Cliem. 184: 313-319.1950.

21. ROBINSON, H. W. and HOGDEN, C. G. The biuretreaction in determination of serum proteins. Jour.Biol. Chem. 135: 727. 1940.

22. SPENCER, D. and WOOD, J. G. The role of molybde-num in nitrate ieductase in higher plants. Aus-tralian Jour. Biol. Sci. 7: 425-434. 1954.

23. STOY, V. Action of different light qutalities on simul-taneous photosynthesis and nitrate assimilation inwheat leaves. Physiol. Plantarum 8: 963-986.1955.

24. STOY, V. Riboflavin catalysed enzyimic photo-reduc-tion of nitrate. Biochim. Biophys. Acta 21: 395-396. 1956.

TRANSLOCATION OF ORGANIC SUBSTANCES IN TREES.I. THE NATURE OF THE SUGARS IN THE SIEVE

TUBE EXUDATE OF TREES'

MARTIN H. ZIMMERMANNCABOT FOUN-DATION, HARVARD UNIVERSITY, PETERSHAM, MASSACHUSETTS

Most investigators regard sucrose as the only formin which carbohydrates are translocated in plants.Some, however, suggest that hexoses are also translo-cated. It is beyond the purpose of this paper to listall these publications, a good bibliography will befound in the review of Crafts (1), and more recentpublications are listed in the work of Ziegler (13).

One major difficulty in translocation work is thefact that possibilities of obtaining material that is ac-tually transported are verv limited. Chemical analy-ses based on tissue extractions yield substances whichoriginate not only from sieve tubes but also fromother types of cells in phloem and non-phloem tissues.There are only two ways of obtaining more or lesspure sieve tube exudate. One method developed re-cently (7, 8) uses cut-off stylets of aphids which werefeeding on the phloem. The other method, tappingthe sieve tube system of trees by cutting into theirbark, was first described by Th. Hartig (4) in 1860.It is this method that has been used in the presentpaper. There is plenty of evidence that the exudateobtained in this way is actually translocated material;Hartig found by microscopic observation that theexudate does come from the sieve tubes. This waslater confirmed by Huber and Rouschal (5). Hartigobserved that a second cut always vields sap if it isapplied above the first one, below the first cut, how-ever, a second cut usually failed to yield exudation(4). Sixty years later, AMunch extended these studiesand found that the sieve tube turgor is released up toseveral meters above a tapping cut (9). Ziegler sup-plied hexoses to the inner bark of trees (Robinia) andsubsequently found them in the exudate below, butnot above the point of application (13). The reasonwe referred to "more or less pure" sieve tube exudateis the possibility that exudate may elute traces of

1 Received December 18, 1956.

impurities from the cut surface (luring the tappingprocess.

Using chromatographic methods, Wanner found in1953 that sucrose is the only sugar in the sieve tubeexudate of Robinia Pseudo-Acacia L. and CarpinusBetulus L. (11). Testing exudate of 10 other Euro-pean tree species, Ziegler also found only sucrose (13).However, in the course of a study on phloem trans-location in white ash (Fraxinus americanta L.), theresults of which will be published elsewhere, it hasbeen found that sucrose is by no means the only sugarin the exudate. It constitutes only 1/10 to 1/5 of thetotal sugar content. The other sugars identified aremembers of the raffinose family: raffinose, stachyoseverbascose. In an attempt to learn whether whiteash is an exceptional case, the sieve tube exudate of16 tree species was collected and analyzed. The re-sults of this survey are presented here.

EXPERIMENTAL

Sieve tube exudate was obtained by cutting intothe inner bark of the tree with a sharp knife. Inolder trees it was necessary to remove the dead barkwith an axe first. The tapping cut must penetratethe region of the functioning sieve tubes, which arelocated a fraction of a millimeter outside the cam-bium. Too deep a cut, however, severs the xylem,which, when it is under negative pressure, will im-mediately draw the exudate back with a hissing sound.The samples were either directly transferred onto thepaper chromatograms or collected in a vial and im-mediately frozen in a Thermos bottle containing anice-salt mixture. Exudate from white ash and redoak was collected from many individual trees duringthe summer and fall of 1955 and 1956. The samplesfrom all other tree species were collectedl during themonth of September 1956.

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THEACTIVITY OFVegetal, Serrano, 113, Madrid, Spain. 3 Present address: New Yorlk State College of Agri-culture, Agricultural Experiment Station, Cornell Uni-versity, Ithaca, New York