Plant and Sol1 X, no.

4 April 1959

THE EFFECT OF MOLYBDENUM AND NITROGEN

DEFICIENCIES ON N I T R A T E R E D U C T I O N
IN PLANT TISSUES *
by E. G. MULDER, R. BOXMA**, and W. L. VAN VEEN
Laboratory of Microbiology, Agriculturai University,
Wageningen, the Netherlands.

INTRODUCTION

The mechanism of nitrate reduction in fungi and higher plants has

been cleared up by N a s o n and E v a n s a s and N i c h o l a s and
N a s o n 11. In experiments with purified cell-free extracts they
found that the nitrate-+nitrite reaction is catalyzed by an enzyme
with either flavin adenine dinucleotide (FAD) or flavin mono-
nucleotide (FMN) as the prosthetic group and moreover molyb-
denum as a constituent. Reduced di- or triphosphopyridine nucleo-
tide (DPNH or TPNH) are required as electron donor. The reduction
sequence was established to be as follows :

T P N H (or DPNH) -+ FAD (or FMN) -+ Mo -2 NO3-

Recent investigations by N i c h o l a s and S c a w i n 12 and by

K i n s k y and M a c E l r o y 4 have shown that nitrate reductase requires
inorganic phosphate for maximal activity. The latter authors
furthermore found that their preparations of nitrate reductase
isolated from Neurospora were able to reduce cytoehrome c in the
presence of TPNH.
In the reduction of nitrite to ammonia a number of enzymatic
reactions are involved. M e d i n a and N i c h o l a s (see N i c h o l a s 9)
have recently identified three metal-containing flavin enzymes in

* The major part of this work was carried out by the authors at the Institute for SOil
Fertility, Groningen.
** Institute for Soil Fertility, C-roningen.

- - 3 3 5 - -
336 E. G. MULDER, R. BOXMA AND W. L. VAN VEEN

cell-free extracts of Neurospora, viz. a nitrite reductase which

reduces nitrite to hyponitrite, a hyponitrite reductase which reduces
hyponitrite to hydroxylamine, and a hydroxylamine reductase
which converts hydroxylamine to ammonia. There is an indication
that the first two of these T P N H - or DPNH-requiring enzymes
depend on the presence of copper or iron, and the third on manga-
nese.
In the present study nitrate-reducing capacities of molybdenum-
deficient cauliflower, spinach, and tomato plants were estimated at
different periods of time after the addition of molybdate to the soil
in which the plants were growing. In a number of experiments the
molybdenum-deficient leaves were infiltrated with dilute solutions
of sodium molybdate.
For comparison, the effect of the addition of nitrate or ammonium
sulphate on the nitrate-reducing capacity of nitrogen-deficient
cauliflower plants, well-supplied with molybdenum, was studied.

EXPERIMENTAL

Protein- and soluble non-protein-nitrogen were d e t e r m i n e d

as d e s c r i b e d ill a p r e v i o u s p a p e r 7.

N i t r a t e was e s t i m a t e d a c c o r d i n g to t h e x y l e n o l m e t h o d 1.

Nitrate-reducing capacity of p l a n t tissues was m e a s u r e d b y u s i n g

coarsely c u t f r a g m e n t s of a p p r o x i m a t e l y 2 t o 3 b y 2 to 3 ram. O n e - g r a m
p o r t i o n s were t r a n s f e r r e d t o T h u n b e r g t u b e s a n d p r o v i d e d w i t h 5 m l 0.06 3/1
p h o s p h a t e b u f f e r (pH 7.1), 1 m l 0.1 3/I m a l i c acid n e u t r a l i z e d w i t h s o d i u m
h y d r o x i d e , 1 m l 0.1 M K N O 3 a n d 2 m l H 2 0 . A f t e r e v a c u a t i o n of t h e t u b e s air
was slowly a d m i t t e d to p r o m o t e t h e u p t a k e of t h e s o l u t i o n b y t h e tissue
f r a g m e n t s . T h e n t h e t u b e s were r e - e v a c u a t e d a n d i n c u b a t e d a t 37°C for h a l f
a n hour. T h e n i t r i t e f o r m e d b y t h e tissue f r a g m e n t s a n d released into t h e
s o l u t i o n was d e t e r m i n e d a c c o r d i n g to t h e G r i e s s - R o m i j n m e t h o d t~ w i t h a
m i x t u r e of ~ - n a p h t h y l a m i n e a n d s u l p h a n i l i c acid.
E n z y m e a c t i v i t y was s t o p p e d b y a d d i n g 1 m l of acetic acid a n d t h e s o l u t i o n
was cleared wittl 3 iill of a s a t u r a t e d s o l u t i o n of (NH4)2SO4. B l a n k values,
o b t a i n e d b y a d d i n g 1 ml of acetic acid a n d 3 m l of s a t u r a t e d (NH4)2SO4 to t h e
c o m p l e t e m i x t u r e before e v a c u a t i o n , were s u b t r a c t e d .
To s t u d y t h e effect of m o l y b d e n m n a n d n i t r a t e s u p p l y to cauliflower,
spinach, a n d t o m a t o p l a n t s on n i t r a t e - r e d u c i n g c a p a c i t y of t h e tissues, t h e
p l a n t s were g r o w n as described in a p r e v i o u s p a p e r 7 oi1 m o l y b d e n u m -
deficient soil e i t h e r in t h e a b s e n c e of m o l y b d e n u m w i t h a d d e d n i t r a t e , or in
t h e p r e s e n c e of m o l y b d e n u m w i t h o u t n i t r a t e . T h e effect of m o l y b d e n u m was
s t u d i e d b y a d d i n g m o l y b d a t e t o t h e soil or b y i n f i l t r a t i n g it i n t o t h e leaves of
 NITRATE REDUCTION IN PLANT TISSUES 33~

m o l y b d e n u m - d e f i c i e n t plants b y v a c u u m infiltration. Similar t r e a t m e n t s

were used for nitrogen in t h e s t u d y of the effect of nitrate.
T h e infiltration of m o l y b d a t e arm n i t r a t e solutions was p e r f o r m e d b y
transferring leaves or leaf fragments to small beakers containing these
solutions which were placed in an exsiccator. A f t e r evacuation, air was
slowly a d m i t t e d and as a result t h e pores of t h e leaf tissues were filled w i t h
liquid. Tile leaves were t h e n exposed to all air s t r e a m to reduce their
moisture c o n t e n t to the original level and i n c u b a t e d for different periods of
t i m e at r o o m t e m p e r a t u r e in a moist atmosphere.

Malic-dehydrogenase activity in leaf tissue was e s t i m a t e d b y

transferring 0.5 g of tissue fragments to T h u n b e r g tubes and adding 1 ml 0.1
M malic acid neutralized w i t h sodium hydroxide, 0.5 m g D P N in 0.5 ml H 2 0 ,
0.2 ml 0.2 ~Ff KCN, 0.5 ml 0.035% m e t h y l e n e blue, and 7.8 mI 0.06 M
p h o s p h a t e buffer (pH 7.0). The T h u n b e r g tubes were e v a c u a t e d before
adding t h e m e t h y l e n e - b l u e solution, air was slowly a d m i t t e d , m e t h y l e n e blue
added, tubes again e v a c u a t e d and i n c u b a t e d at 37°C. T i m e of reduction of
m e t h y l e n e blue was t a k e n as a measure o f malic-dehydrogenase a c t i v i t y a

C a t a l a s e a c t i v i t y was e s t i m a t e d as %llows. 0.2 to 0.5 g of ground p l a n t

m a t e r i a l was transferred w i t h 10 ml of 1/15 M p h o s p h a t e buffer, p H 7.0, to a
I-1 E r l e n m e y e r flask fitted w i t h a small, calibrated s e p a r a t o r y funnel con-
taining a 6% solution of H20~ ill a similar p h o s p h a t e buffer. The flask was
further connected with a calibrated " B u n t e " burette, filled w i t h H20. Ten ml
of H202 solution was added to the m a c e r a t e while the flask was in connection
with t h e a t m o s p h e r e via the t h r e e - w a y stopcock of the burette.
I m m e d i a t e l y after m i x i n g the H~O2 and the p l a n t extract, t h e stopcock
was t u r n e d so t h a t the a m o u n t of O2 evolved b y t h e eatalase a c t i v i t y could be
nleasured b y the displacement of w a t e r from t h e burette. Care was t a k e n t h a t
t h e readings were m a d e at atmospheric pressure in the E r l e n m e y e r flask.
D u r i n g the test the flask was shaken constantly.

RESULTS

1. E//ect o~ molybdenum, added to molybdenum-de/icie~l cauli/lower,

spinach, and tomato ibla~¢ts, on ~Ktrate-reduci~¢g capacity o~ these
plau~s
a) N i t r a t e r e d u c t i o n in m o l y b d e n u m - d e f i c i e n t p l a n t s
u p o n a d d i t i o n of Na2Mo04 to t h e soil.
C a u l i f l o w e r p l a n t s . Estimations of nitrate-reducing capacity
were carried out in leaf fragments of molybdenum-deficient plants
approximately 4~, 9~, and 24}hours after application of 2.5 mg molyb-
date to 1 kg of the molybdenum-deficientsoil in which the plants were
growing. As will be seen from Table I, A, a twentyfold increase of ni-
trate-reducing capacity was found after 4} hours. After an incubation
538 E.G. MULDER, R. BOX?vIA AND W. L. VAN V E E N

TABLE I

Effect of added molybdenum on nitrate-reducing capacity of molybdenum-

deficient )iants.*
N0a- N03-
Treatment of Plant reducing Treatment of Plant reducing
plants tissue capacity plants tissue capacity

A 0 (continued)
Cauliflower 0Mo, 1 dayMo Stem 4.0
0 Mo, control Leaf 0.3 Seed leaf 2.3
Stem 0.4 0 Mo, 3 days Mo Leaf 3.4
0 Mo, 4-} h Mo Leaf 6.3 Stem 5.2
Stem 2.0 Seed leaf 3.0
0 Mo, 9} h Mo Leaf 7.0 0 Mo, 6 days No Leaf 0.9
Stem 2.7 Stem 2.8
OMo, 2 4 } h M o Leaf 4.7 Seed leaf 1.1
Stem 2.7 + Mo, control Leaf 0.5
+ Mo, control Leaf 3.1 Stem 1.2
Stem 2.0 Seed leaf 0.7
B D
Cauliflower Tomato
0 Mo, } h H~O Leaf 1.C 0 Mo, control Leaf 0.2
0 Mo, 2 h Mo Leaf 2.9 Stem 1.3
0 IV[o, 2 h H20 Leaf 1.1 0Mo, 1 d a y M o Leaf 2. l
0 Mo, 4~} h No Leaf 6.3 Stem 2.4
0 Mo, 4~ h H20 Leaf 1.3 0 Mo, 2 days Mo Leaf 1.2
Stem 2.4
13 0 No, 5 days Mo Leaf 1.2
Spinach Stem 1.8
0 Mo, control Leaf O.C 0 Mo, 7 days No Leaf 0.5
Stem 0.5 Stem 2.4
Seed leaf 0.5
OMo, I d a y M o Leaf 3.7

* Averages values for protein-N, soluble non-protein-N (except nitrate-N), and ni-
trate-N, in % of dry matter, respectively, of:
Cauliflower: A, 0 Mo control, leaf: 3.2, 1.7, 2.2; stem: 1.5, 1.I, 3.9; + Mo control,
leaf: 4.4, 1.4, 1.1; stem: 1.5, 1.1, 3.3. B, 0 Mo, H20, leaf: 3.3, 1.8, 2.4.
Spinach: 0 Mo control, leaf: 2.8, 1.6, 3.1; stem: 1.9, 1.4, 3.4; seed leaf: 2.3, 1.1, 3.0; +
Mo control, leaf: 4.5, 1.6, 0.7; stem: 2.3, 1.5, 2.7; seed leaf: 2.8, 1.4, 1.5; 3 and 6 days after
treatment NOs-N of the leaves had decreased to 2.7 and 1.8% respectively.
Tomato: 0 Mo control, leaf: 3.9, 0.9, 2.3; stem: 1.6, 0.6, 3.8; 2, 5 and 7 days after Mo
treatment NOa-N of the leaves had decreased to 1.9, 0.7, and 0.5% respectively.
** Averages of duplicate values expressed as/~g NaNO2 formed per }h per mg protein.

time of 9~ h some further increase of nitrate-reducing power had

occurred. After 24 hours considerably lower values were found in
leaf fragments, but not in stem tissue.
In a subsequent experiment with cauliflower plants nitrate-
 N I T R A T E R E D U C T I O N IN P L A N T T I S S U E S 339

reductase activity was measured approximately two hours after the

addition of molybdate to the soil in which the molybdenum-deficient
plants were growing. In this case nitrate-reducing capacity of leaf
fragments had increased to a threefold level of the control (Table I,
B). Plants of the same series tested five hours after the addition of
molybdenum, showed a much higher activity. Apparently under the
conditions of these experiments (greenhouse, autumn, approxi-
mately 20°C) nitrate-reducing capacity in molybdenum-deficient
cauliflower leaves reached its full activity 4 to 5 hours after the
addition of molybdate to the soil in which the plants were growing.
S p i n a c h p l a n t s . Estimations of nitrate-reducing capacity in
leaf and stem fragments were carried out I, 3, and 6 days after the
application of molybdate. High values were observed 1 day after
molybdenum treatment (Table I, C). Six days after the addition of
molybdenum, the nitrate-reducing capacity of molybdenum-
deficient plants had decreased to a level slightly higher than that of
plants supplied with adequate amounts of molybdenum from the
beginning of their development.
The molybdenum-deficient spinach plants were extremely rich
in nitrate nitrogen; the highest value found, viz. 3.45°/0, corresponds
with a nitrate content of approximately 25~o of the dry matter.
Three day-s after the addition of molybdenum, the nitrate contents
of the leaves and seed leaves had clearly decreased whereas the
protein values had increased.
T o m a t o p l a n t s . Two experiments with molybdenum-deficient
tomato plants have been carried out. In the first one it was shown
that 4½ hours after the addition of molybdate to the soil, it had been
taken up by the plants and had affected a pronounced rise in
the nitrate-reducing capacity of the leaves.
In the second experiment, carried out in the second part of
August 1954, nitrate-reductase activity was determined in leaves,
stems and roots, 1, 2, 5, and 7 days after the addition of molyb-
denum. The highest value for leaf tissue was found 1 day after the
application of molybdenum. After longer incubation times lower
values were found (Table I, D). These results are in agreement with
those of the preceding experiment with spinach plants. In stem
tissue such variation did not occur.
340 g . G. MULDER, R. BOX1KA AND W. L. VAN VEEN

b) N i t r a t e r e d u c t i o n in m o l y b d e n u m - d e f i c i e n t plants
upon infiltration with sodium molybdate.
C a u l i f l o w e r p l a n t s . One half of molybdenum-deficient leaves
was infiltrated with a solution containing 25 ppm of Na2MoO4.2H20,
the other half with H20. It was shown that two hours after com-
pletion of the infiltration with molybdate, the nitrate reducing
capacity had reached its maximal value (Table II, A).
S p i n a c h p l a n t s . A comparison was made between nitrate-

TABLE II

Effect of infiltrated molybdate on nitrate reduction in Mo-deficieut cauliflower,

spinach, and tomato leaves.*
Nitrate- Nitrate-
Treatment of plants Ineu- reducin~ Treatment of plants Incu- reducin~
and infiltrated bation capacity and infiltrated bation capacity
solution time, h ** solution time, h **

A B (Spinach continued'
Cauliflower q- Mo, HlO 1 3.1
0 Mo, 1120 0.4 q- Mo, HlO 4.25 4.1
0 Mo, Na2MoO4 5.8 n- Mo, Na2Mo04 1 3.3
0 Mo, Na~MoO4 4.2 + Mo, Na~Mo04 4.25 4.5

B G
Spinach Tomato
0 Mo, no infil- 0 Mo, H20 0.4
tration 0 0.6 0 Mo, Na2IKoO4 2.4
0 Mo, H20 1 2.2 0 Mo, Hi0 0.4
0 No, H20 4.25 3.4 0 Mo, Na2MoO4 4.9
0 Mo, Na2MoO4 1 4.3
0 Mo, Na2MoO4 4.25 8.4
q- Mo, no infil-
tration 3.5

* Average values for protein-N, soluble non-protein-N (except uitrate-N), and nitrate-
N, respectively, in % of dry matter: Cauliflower: 2.9, 1.4, 2.3. Spinach, 0 Mo: 3.2, 1.5,
2.3; + Mo: 4.3, 1.5, 0.7. Tomato: 3.3, 1.2, 2.2.
** Averages of duplicate values expressed as #g NaNO2 formed per ½ h per mg protein..

reducing capacities of molybdenum-deficient leaves infiltrated with

a molybdate-containing solution (I ml = 20 ag of Na2MoO4.2H20)
and H20, respectively. Estimations of nitrate-reducing capacities
were made 1 and 4} h after infiltration. The results of this experi-
ment recorded in Table II, B, demonstrate that 1 hour after com-
pletion of the infiltration with sodium molybdate, nitrate-reducing
capacity has been restored only partly. At 4} h, a value was obtained
 N I T R A T E R E D U C T I O N IN P L A N T T I S S U E S 341

which was considerably higher than that of normal plants, even

when the latter had been infiltrated with molybdate.
Infiltration with H 2 0 alone increased nitrate reduction of molyb-
denum-deficient plants considerably. In the case of normal plants
this H20-effect was also observed, but to a smaller extent.
T o m a t o p l a n t s . Coarsely cut leaf fragments of molybdenum-
deficient plants were infiltrated with H~O or a solution containing
20 ppm of Na2MoO4.2H20. Two and four hours, respectively, after
completion of the infiltration, the leaf fragments were transferred to
Thunberg tubes and nitrate-reducing capacities estimated. Two
hours after the infiltration with molybdate, the molybdenum-
deficient leaf fragments had not yet obtained their maximal nitrate-
reducing capacity (see Table II, C).

2. Effect o~ the nitrogen supply to cauli/lower, spinach, and tomato

plants on nitrate-reducing capacity
Since nitrate reductase presumably is an adaptive enzyme, it was
suggested that plants inadequately supplied with nitrate nitrogen
would possess a lower nitrate-reducing capacity than those well
supplied with this nutrient. Experiments were therefore done with
nitrogen-deficient plants which had been supplied, either b y appli-
cation to the soil, or b y infiltration, with nitrate or an ammonium
compound.
a) N i t r a t e r e d u c t i o n in n i t r o g e n - d e f i c i e n t p l a n t s u p o n
a d d i t i o n of K N 0 3 t o t h e soil. Experiments were carried out with
spinach and tomato plants. These plants were grown in similar
Neubauer jars as used in experiments with molybdenum, while the
same molybdenum-deficient soil was employed. This soil was also
dressed in the same way except that 2} mg Na2Mo04 was given per
1 kg of soil, and nitrate was omitted.
In the case of spinach, the nitrogen-deficient plants were charac-
terized b y a much reduced development as compared with plants
normally supplied with nitrate; their leaf colour was, however, only
of a slightly lighter green. 0,75 g KNOa was added per pot on 5
October 1954 and harvests were made after 1, 2, 3, and 6 days. In
addition to nitrate-reducing capacity, estimations were made of
protein-N, soluble non-protein-N and nitrate-N. The results of these
experiments are recorded in Table III, A, those of a similar experi-
ment with tomato plants in Table III, B. It will be seen that ad-
342 E . G . MULDER, R. BOXMA AND W. L. VAN V E E N

TABLE I I I

Effect of added KNO3 on nitrate-reducing capacity of nitrogen-deficient spinach

and tomato plants.*
[ NO3- NO~-
Treatment of plants Plant reducing Treatment of plants Plant reducing
tissue capacity tissue capacity

A B
spinach Tomato
0 N, control Lear 0.8 0 N, control Leaf 0.0
Stem 2.1 Stem 3. l
Seed leaf 0.1 0 N, 1 day KNOa Leaf 0.8
i
0 N, 2 days KNOg i Leaf 4.3 Stem 7.6
I Stein 3.7 0 N, 2 days KNOB Leaf 0.4
Seed leaf 2.5 Stem 4.1
0 N, 3 days KNOa [
Leaf 3.3 0 N, 5 days KNOa Leaf 1.9
] Stem 3.9 Stem 3.4
Seed leaf 1.9 0 N, 7 days KNOa Leaf 1.I
0 N, 6 days KNOa Leaf 2.1 Stem 2.0
Stem 2.8
: Seed leaf 1.6
+ N, control 1 Leaf 0.9
I Stem 2.7
Seed leaf 0.5

* Average values for protein-N, soluble non-protein-N (except nitrate-N), and ni-
trate-N, respectively, in. % of dry matter:
spirmch: 0 N control, leaf 3.7, 1.3, 0.l; stem 2.2, 1.7 0.0; seed leaf 1.8, 0.9, 0.1; 2
and 6 days after KNOa treatment NOa-N in the 1eaves increased to 0.6 and 0.7~0
respectively.
Tomato: 0 N control, leaf 1.9, 0.3, 0.0; stem 0.7, 0.5, 0.0; 2 and 7 days after KNO3
treatment NOa-N in the leaves had increased to 0.4 and 0.7% respectively.
** Averages of duplicate values expressed as [zg NAN02 formed per } h per mg of
protein.

dition of nitrate to nitrogen-deficient plants brought about a pro-

nounced rise of nitrate-reducing capacity. As with the molybdenum
treatment of molybdenum-deficient plants this effect was most
pronounced shortly after the application of the nitrate. After 6 or 7
days it was clearly reduced.
b) E f f e c t of a d d e d n i t r a t e a n d a m m o n i u m s u l p h a t e on
n i t r a t e - r e d u c i n g c a p a c i t y of n i t r o g e n - d e f i c i e n t c a u l i -
f l o w e r l e a v e s (28 October i954). The nitrogen-deficient plants
were considerably smaller than those dressed with nitrate, their leaf
colour was of a slightly lighter green than normal, while the seed
 N I T R A T E R E D U C T I O N IN PLANT TISSUES 343

leaves had died. Ten pots of this series were dressed with 5 ml of a
solution containing I00 mg KN03 per ml. This solution was washed
into the soil with 20 ml H20. A second series of ten pots was dressed
with an equal amount of nitrogen in the form of (NH@ 2SO4, whereas
a third series received H~O only. At different periods of time after
the addition of the nitrogenous compounds, plants were harvested
and tested for nitrate-reducing capacity. The results of this experi-
ment are recorded in Table IV. It will be seen that 4½ h after the

TABLE IV

Effect of added KNOa and (NH4)2SO4 on nitrate-reducing capacity of nitrogen-

deficient cauliflower leaves *
Ineu- NOa- Incu- NOa-
bation reducing bation reducing
Treatment of plants time, capacity Treatment of plants time, capacity
hours ** hours **
H~O, control 4.5 1.6 (NH4) 2SO4 23 2.7
KNOa 4.5 4.3 tI20, control 47 1.3
(NH~)~SO4 4.5 3.2 KNOB 47 6.4
H20, control 23 0.7 (NH4)2SO4 47 3.4
KNOs

* Average values for protein-N, soluble non-protein-N (except nitrate-N), and ni-
trate-N, respectively, in % of dry matter: 2.0, 0.7 and 0.0.
** Averages of duplicate values expressed as /~g NaNO2 formed per ½ h per mg
protein.

application of nitrate, the nitrate-reducing capacity of the leaves had

considerably increased. After 1 and 2 days, respectively, this activity
had stilI further increased. Plants dressed with ammonium sulphate
also showed increased nitrate reduction though to a lesser extent
than those treated with nitrate.
c) I n f i l t r a t i o n of KN03 i n t o n i t r o g e n - d e f i c i e n t t o m a t o
l e a v e s (1 November 1954). For this experiment plants with
moderate symptoms of nitrogen deficiency were employed. Coarsely
cut leaf fragments were infiltrated with a KNO3-containing solution
(1 ml = 10 mg) and H20, respectively. 22/3 and 5 h after infiltration,
nitrate-reducing capacities were determined. As may be seen from
the results in Table V, A, nitrate-reducing power had considerably
increased 22/3 h after infiltration with a nitrate-containing solution.
After 5 h a further increase had taken pIace.
344 E.G. M U L D E R , R. BOXMA A N D W. L. VAN V E E N

d) E f f e c t of i n f i l t r a t e d KNOa, (NH4)2S04 a n d K2S04 on

n i t r a t e - r e d u c i n g c a p a c i t y of n i t r o g e n - d e f i c i e n t t o m a t o
p l a n t s . In order to investigate whether the nitrate-reducing
TABLE V
Effect of infiltrated KNOB and (NH4)2804 on nitrate-reduction ilX nitrogen-
deficient tomato leaves.
t Inca- Nitrate- ]~nca- Nitrate-
T r e a t m e n t of T r e a t m e n t of
bation j reducing bation reducing
plants and in- plants and ill-
time, . capacity time, capacity
fiItrated solution filtrated solution
hours [ :'~* hours
A* L B
0 N, HeO i 3 1.6 0 N, H~O 3~ 26
0 N, KNOa 2~ 2.9 0 N, KNOa 3½ 129
0 N, H~O 5 1.0 0 N, (NH4)2SO4 3½ 16
0 N, KNOa 5 4.1 0 N, K~S04 3½ 20
* Average values for protein-N, soIuble non-protein-N (except nitrate-N), and ni-
trate-N, respectively, in % of dry m a t t e r : 3.2, 0.8, 0.0; after infiltration NOa-N had
increased to 0.6%.
** Averages of duplicate values expressed as fig NaNO2 formed per ½h per m g protein.
*** Averages of duplicate values expressed as fig NaNO.2 formed per ½ h per g fresh
weight.

capacity could be restored by infiltration of the leaves with solutions

of KNOa or (NH4)~S04, an experiment was carried out with N-
deficient tomato leaves on 5 November 1954. The infiltrations were
done with solutions of KNOa (1 ml = 10 mg), (NH4)2S04 (1 ml =
6.5 rag) and K2S04 (1 ml = 8.6 rag). The temperature during the
incubation was 22°C. Five hours after the completion of the infil-
tration, the leaves were cut into coarse fragments and nitrate-
reducing capacity was estimated. As will be seen from the data of
Table V, B, nitrate-reducing capacity was restored only if the leaves
had been infiltrated with KNOa.
That the effect of infiltrated KNOa, in the preceding experi-
ments, was due to the formation of an adaptive nitrate-reducing
enzyme and not to an improved nitrate supply to an existing enzyme
system, was concluded from an experiment using leaves of normal
cauliflower plants grown in the presence of nitrate. These dark-green
leaves contained no detectable nitrate and were assumed to have a
normal enzyme content. Fragments of such leaves produced
immediately - in an incubation period of half an hour - considerable
quantities of nitrite when incubated in evacuated Thunberg systems
in the presence of nitrate. Controls in the absence of added nitrate
produced little nitrite and hence it must be concluded that this
 N I T R A T E R E D U C T I O N IN PLANT TISSUES 345

substance originated by reduction of added nitrate. The absence of a

lag phase in the development of reducing power suggests that
nitrate reductase was initially present in these leaves grown on
nitrate, whereas the delay in development of nitrate reduction in
leaves grown in the absence of nitrate in previous experiments
suggests adaptive enzyme formation.
e) C o m p a r i s o n of t h e e f f e c t s of i n f i l t r a t e d KNOs on N-
d e f i c i e n t a n d of i n f i l t r a t e d Na2Mo04 on m o l y b d e n u m -
d e f i c i e n t t o m a t o p l a n t s . For this experiment tomato plants
were grown in molybdenum-deficient soil a) with added molyb-
denum b u t without nitrate, b) with added KNOa but without
molybdenum. The symptoms of nitrogen deficiency were less
severe than those of molybdenum deficiency. The leaves of both
series were harvested at 10 a.m. on 2 November 1954. The nitrogen-
deficient samples were infiltrated with a solution of KNOa (1 ml ---=
10 mg); those from molybdenum-deficient plants were infiltrated
with a solution containing 20 ppm Na2MoO4.2H20. Control samples
of both series were infiltrated with H20 only. Nitrate-reducing
capacities of coarsely cut leaf fragments were estimated b y the usual
procedure 2, 4, and 6 hours after infiltration (Table VI).
TABLE VI
Nitrate-reducing capacities of nitrogewdeficient tomato leaves * infiltrated with
KNOa, and moIybdenum-deficient tomato leaves * infiltrated with Na2MoO4.2HlO
Incu- Solutions in- Ineu-
Solutions in- bation Nitrate- filtrated in bation Nitrate-
filtrated in N- time, reducing Mo-deficietlt time, reducing
deficient leaves hours capacity ** leaves hours capacity **
H~O 2 0.9 H~0 2 0.1
KNOa 2 2.6 Na2MoOa 2 1.0
H20 4½ 1.8 H20 4½ 0.1
KN0a 4} 6.3 Na2MoO4 4½ 2.8
H20 6½ 1.1 H~O 6½ 0.3
KN0a 6½ 5.8 Na2MoO4 6½ 3.4
* Average values for protein-N, soluble non-protein-N (except nitrate-N), arid m-
trate-N, respectively, in % of dry matter: 0 N: 3.1, 0.9, 0.0; after infiltration NOa-N
had increased to 0.5%. 0 Mo: 3.1, 1.4, and 2.6%.
** Averages of duplicate values expressed as # g NAN02 formed per ½ 1! per mg of
protein.

From the results in Table VI it m a y be seen that two hours after

completion of the infiltratiorr the nitrate-reducing capacities of N-
deficient plants had been restored to approximately 50 per cent and
that of Mo-deficient plants to approximately 30 per cent.
346 E. G. M U L D E R , R. B O X M A A N D W . L. V A N V E E N

3. Other/actors affecting nitrate-reducing capacity in lea~/ragments

a) E f f e c t of c u t t i n g a n d g r i n d i n g of l e a v e s on n i t r a t e
r e d u c t i o n. To investigate whether the nitrite formation observed
in leaf fragments in the preceding experiments must be attributed
to intact or disruptured cells, experiments were carried out with leaf
fragments of different sizes and with ground tissue. In the first
experiment, spinach plants, grown in the presence of both nitrate
and motybdate, were used. Leaves from these plants were cut into
fragments of approximately 1 by 1,2 b y 2 and 5 b y 5 ram, respective-
ly, and tested for nitrate-reducing capacity as described above. The
highest values were found in coarsely cut fragments, viz 52 #g
NAN02 formed per I g fresh tissue per ½ h at 37°C; fragments of
2 mm had formed 39 #g NaNO2 under the same conditions and those
of 1 mm only 15 ~g. These results demonstrate that nitrate reduction
in leaf fragments takes place mainly in undamaged cells. This was
still more clearly shown when the leaf tissue was ground in a mortar.
In the latter experiment a comparison was made between fragments
cut to different sizes from normal cauliflower leaves and a macerate
of the same amount of plant material. To investigate the possibility
of nitrite binding or nitrite reduction, two samples of ground leaf
tissue were provided with a certain amount of nitrite before evacu-
ating the Thunberg tubes. The results of this experiment, recorded
in Table vii, provide evidence that in addition to inactivation of
the nitrate-reducing enzyme system, loss of nitrite occurs in leaf
macerates. Nevertheiess extracts of ground leaf tissue, infiltrated

TABLE VII

E f f e c t of c u t t i n g a n d g r i n d i n g of t o m a t o l e a v e s on n i t r a t e - r e d u c i n g c a p a c i t y a n d
nitrite removal.
Nitrate-reducing
TreatmeIlt of leaf tissue
capacity *
1. Whole l e a v e s . . . . . . . . . . . . . . . . . . . 51
2. F r a g m e n t s of 5 b y 5 m m . . . . . . . . . . . . . . 65
3. F r a g m e n t s of 1-2 b y 1-2 m m . . . . . . . . . . . . 22
4. Leaf m a c e r a t e . . . . . . . . . . . . . . . . . . 8
5. L e a f m a c e r a t e + 70/~g N a N O e . . . . . . . . . . . 51
6. W a s h e d residue of m a c e r a t e . . . . . . . . . . . . 10
7. F r a g m e u t s of 5 b y 5 m m , i n f i l t r a t e d w i t h leachings of
treatment 6 . . . . . . . . . . . . . , . . . . . 54

* A v e r a g e s of d u p l i c a t e v a l u e s e x p r e s s e d as # g N a N O 2 f o r m e d per ½ h p e r ½ g fresh
tissue.
 N I T R A T E R E D U C T I O N IN P L A N T T I S S U E S 347

into leaf fragments, hardly affected their nitrate-reducing power.

Hence the depressing effect of grinding on nitrate reduction is not
due to the presence of inhibitory substances.
The disappearance of nitrite in ground leaf tissue was shown more
clearly in an experiment with spinach. Five grams of leaf macerate
were provided with 300 #g NAN02 in addition to malic acid and
phosphate buffer in the usual concentration. The mixture was
incubated in an evacuated Thunberg tube for 1/4 h at 37°C and
subsequently analysed for nitrite. Only 122 #g of N a N Q were found
which indicates a loss of approximately 60 per cent of the added
nitrite.
The effect of ground leaf tissue on nitrite apparently is partly
chemical and partly enzymatic. This was concluded from an
experiment in which a comparison was made between fresh and
boiled samples of a macerate of cauliflower leaves. 0.7 g of this
macerate supplied with 250 !,g of NaNO2 under the usual conditions
brought about a loss of 139 yg of NaNO2 in ½ h at 37°C when used in
the fresh state and of 103 #g when it had been boiled. It is also
possible that the effect of boiling is due to loss of nitrite-binding by
the protein.
b) E f f e c t of a d d e d y e a s t e x t r a c t o n n i t r a t e r e d u c t i o n in
! e a f m a c e r a t e . If it is assumed that nitrate reduction in leaf tissue
is brought about b y the activity of nitrate reductase, it can be
suggested that either desorganization of the enzyme system or lack
of substrate ( D P N H or TPNH) were responsible for the inactivation
of the nitrate-reducing capacity in disruptured cells. Therefore a
few experiments with yeast extract (which is a source of FAD) have
been carried out.
For the preparation of yeast extract, 2 5 g of dried "Engedura"-
yeast were rubbed in a mortar, dissolved in 100 ml boiling water, and
boiled for a few seconds. After cooling, the solution was centrifuged
and the clear yellow centrifugate used for the enzyme tests.
In the first two experiments with macerates of normal spinach
leaves a remarkable effect of yeast extract on restoring nitrate
reduction was observed (see Table VIII).
In the case of leaf fragments, yeast extract had no effect on
nitrate reduction.
In subsequent experiments with spinach and cauliflower leaves
the effect of added yeast extract was considerably less pronounced
348 E.G. MULDER, R. B O X M A A N D W . L. V A N V E E N

TABLE VIII

Effect of yeast extract on nitrate-reducing capacity of ~ o u n d spillach leaves

First experiment Second experiment
NO~- NO~-
Treatment reducing Treatment reducing
capacity* capacity*
0.5 g leaf macerate 3 0.5 g coarsely cut leaf f r a g m . 61
0.5 g I.m. + 1 m l y e a s t ex- dito + 1 mI yeast extract 65
t r a c t , added before grinding 30 0.5 g Ieaf macerate 4
0.5 g 1.m. + 1 m1 y e a s t ex- dito + } m l yeast extract 10
t r a c t added 20 min after dito + 1 mI yeast extract 33
grinding 19 dito + 2 m i yeast extract 24
1 m l yeast extract 4 dito + 51 # g N A N 0 2 22
0.5 g 1.m. + 6 0 / ~ g N a N O 2 29 1 m l yeast extract 3
2 m l yeast extract 4

* Averages of duplicate values expressed as/~g N a N O e formed per ½ hour ½ g fresh

tissue at 37°C.

than in the above-mentioned two tests. This was presumably due to

the fact that yeast extract itself caused a loss of added nitrite (2 ml
yeast extract added to the usual reaction mixture in evacuated
Thunberg tubes at 37°C reduced added nitrite in ½ h from 130 to 30
/Ag. This was not an enzymatic effect since it was also exerted by
yeast extract which had been boiled twice.
When a correction was made for the nitrite-decomposing effect of
both leaf macerate and yeast extract, a small but consistent nitrate
reduction was observed in the leaf-macerate-yeast-extract mixtures
of the later experiments.
c) E f f e c t of i l l u m i n a t i o n on n i t r a t e - r e d u c i n g c a p a c i t y .
A comparison was made between cauliflower plants kept in the dark
or illuminated, for 3 hours, in a thermostate of approximately 20°C.
No difference in nitrate-reducing capacity of leaf fragments was
observed.
In a subsequent experiment, cauliflower plants, kept for two days
in the dark at 20°C, showed a somewhat reduced nitrate-reducing
capacity as compared with that of plants kept under greenhouse
conditions (27 and 32 #,g of NaNO~ formed per 1 g of leaf fragments
per ½ h, respectively). When the plants were kept first in the dark
for one day and then exposed to light in the greenhouse for one day
an increased nitrite production was found, viz 45 ¢,g NaNO2 per 1 g
of leaf fragments.
 N I T R A T E R E D U C T I O N IN P L A N T T I S S U E S 349

4. Effect o~ molybdenum and nitrogen supply o~ cauliflower, spinach,

and tomato plants o1¢malic-dehy#ogenase activity
If it is assumed that D P N H or T P N H is used as the substrate in
nitrate reduction, then a dehydrogenase activity is required to keep
these nucleotides in the reduced state. The importance of dehydro-
genase activity for nitrate reduction in leaf fragments of tomato
plants was demonstrated in an experiment in which malic acid was
omitted from the usual reaction mixture. Thirty-three Fg of NAN02
were formed per ½g of leaf fragments against 47 #g when 1 ml 0.1 M
malic acid had been added and 43 Fg in the presence of 1 ml 0.1 M
succinic acid. The fact that both malic and succinic acid affected
nitrite formation, indicates that both malic and succinic dehydro-
genases were active.
Malic-dehydrogenase activity was estimated according to the
above-mentioned method in a number of tissues which were also
tested for nitrate-reducing capacity. Table IX gives the results of

T A B L E IX

Effect of molybdenum supply to spinach and cauliflower plants on malic-dehy-

drogenase and nitrate-reductase activities
Spinach leaves Cauliflower leaves
Nitrate- Nitrate-
Treatment Nalic dehy- redue- Treatment IKalic dehy- redue-
drogenase * drogenase *
tase ** tase **
0 Mo 35 (57) 9 0 Mo 6~ (8I) 15
0Mo, 5 h 0 Mo, 2 h
after addition after addition
of Na2MoO4 s3 (7s) 74 of NazNIoO4 31 01oo) 42
+ 3{0 37 (53) 60 0 Mo s6 ()2oo) 17
0 Mo, 4½ h
Na2~foO4 53 ()14o) 99
0 Mo 70 012o) 18

* Averages of duplicate values. A c t i v i t y expressed as time (in minutes) required for

complete reduction of added methylene blue by ½ g leaf fragments. In parentheses~
values obtained when KCN was omitted from the reaction mixture.
** Averages of duplicate values expressed as /zg NaNO~ formed per } h per g fresh
tissue.

two experiments in which the effect of molybdenum supply on

malic-dehydrogenase activity was estimated. For comparison
nitrate-reducing capacities are recorded. It will be seen that in the
case of spinach leaves no difference in malic-dehydrogenase activity
350 E. G. MULDER, R. BOXMA AND W. L. VAN V E E N

was observed between molybdenum-deficient and normal leaf

tissue, while added molybdate had a depressing effect on activity. In
cauliflower leaves, however, added molybdenum clearly promoted
malic-dehydrogenase activity.
In the case of nitrogen-deficient cauliflower, added nitrate which
promoted nitrate-reducing capacity (cf Table IV) did not affect
malic dehydrogenase. Addition of (NH4) 2S04 which favoured nitrate
reduction slightly, adversely affected malic dehydrogenase (Table X).
TABLE X

Effect of added KN03 and (NH4)~SO~ on malic~dehydrogenase activity of cauli-

flower leaves.*
Treatment Malic dehy~ Treatment Malic dehy~
drogenase ** drogenase **
ON 14 (>90)
0 N , 4½h KNOa 13 09o) 0 N, 4½ h (NH@2S04 3o ()90)
ON 12 (I 17)
0N, 2 3 h K N O a 12 (10s) 0 N, 23 h (NH4)2SO4 21 (138)
ON 11 0100)
0N, 47hKNOa 14 (>i00) 0 N, 47 h (NH4)2SO4 29 01oo)

* Nitrate-reducing capacities in Table IV

** Time (in minutes) required for complete reduction of added methylene blue; in
parentheses: values obtained without KCN. Averages of duplicate values.

5. E//ect o/ molybdenum supply on catalase activity o/ cauliflower,

spinach, and tomato plants
a) C o m p a r i s o n of m o l y b d e n u m - d e f i c i e n t a n d n o r m a l
c a u l i f l o w e r a n d s p i n a c h l e a v e s . Readings were made after
different periods of time (see Fig. 1). It will be seen that catalase
activity of molybdenum-deficient plants was considerably less than
that of normal plants. As a result of this effect, pronounced differ-
ences in behaviour of molybdenum-deficient and normal plants were
sometimes obtained when these plants were placed in dilute
solutions of H202. The former lost their turgescence within a short
time whereas normal plants were not affected. These results were
not consistent, however; in a number of cases, molybdenum-
deficient spinach and tomato plants were not affected by H202.
b) E f f e c t of a d d e d r n o l y b d a t e . In a few further experiments
the effect of added molybdate on catalase activity of molybdenum-
deficient spinach and tomato plants was investigated. Although
normal leaf tissue had a considerably higher catalase activity than
 N I T R A T E R E D U C T I O N IN PLANT T I S S U E S 351

zx
rn{ O~ per mg p r o t e i n

j zx

0 Mocauliflower leaves
+ Mo ,,
0 0 Mo spinach ,,
---1- + M o

7 ~

. . . . . . . . " .........

I ju~ o ........
. . . . 1 I I I
30 60 90 120

Fig. 1. The effect of molybdenum supply on the catalase activity (ml 02 per
mg protein) of cauliflower and spinach leaves. + Mo = 2.5 mg Na2MoO4.2H.20
per pot, Averages of quadruplicate values.

molybdenum-deficient, the effect of added molybdate, at least

during the first days after addition, was slight. In contrast to
nitrate-reducing capacity of Mo-deficient plants which was always
restored within one day after molybdenum treatment, catalase
activity six days after molybdenum treatment had not yet reached
the value of normal plants.

DISCUSSION

The effect of molybdenum on nitrate reduction in cauliflower,

spinach, and tomato leaves was studied by adding molybdate to
Mo-deficient plants or by infiltrating leaves of the latter plants with
molybdate-containing solutions. Nitrate-reducing capacities of the
352 E.G. MULDER, R. BOXMA AND W.L. VAN VEEN

tissues were determined under anaerobic conditions at 37°C * at

different periods of time after molybdate had been provided;
malate served as hydrogen donor. Under these conditions relatively
large amounts of nitrite were formed and released into the solution
in which the tissue fragments were suspended. Since omission of
malic acid from the reaction solution reduced nitrite formation,
while added D P N H had no promoting effect, it was assumed that e-
lectron donors (DPNH or TPNH) are available or are produced in the
tissue fragments in adequate amounts for optimal nitrate reduction.
Under the conditions of the experiments, it took approximately
4 hours to restore nitrate-reducing capacity to molybdenum-
deficient leaf tissue by the addition of sodium molybdate. It was
remarkable that in most cases the nitrate-reducing capacities of
Mo-deficient tissues provided with molybdate were higher than those
of normal tissues well supplied with molybdenum from the begin-
ning of their development. This indicates the accumulation of some
stimulating compound in molybdenum-deficient plants.
The experiments with nitrogen-deficient cauliflower, spinach, and
tomato plants provided evidence for the fact that nitrate reductase
in these plants is an adaptive enzyme. The beneficial effect of
ammonium sulphate on the nitrate reduction of nitrogen-deficient
cauliflower plants, observed 4½ hours after its addition to the soil
(Table IV) m a y have been due either to a very rapid nitrification of
part of the added (NH4)2S04 or to the presence of some nitrate
impurities in the ammonium=sulphate solution. That (NH4)2SO4
itself has no effect on the nitrate-reducing capacity was concluded
from the infiltration experiment recorded in Table V. These results
are in agreement with those of C a n d e l a et al. ~ who found in cauli-
flower plants grown with ammonium sulphate as the nitrogen source,
values for nitrate-reductase activity one-third and one-sixth of those
observed in plants given nitrate or nitrite. N i c h o l a s and N a s o n 11
obtained similar results with fungi.
The fact that a few hours after the addition of nitrate to the soil,
the nitrate-reducing capacity of the leaves had increased to a large
extent, makes it highly improbable that the restoration of the
enzyme activity was due to the synthesis of entirely new protein
from assimilated nitrate.

* A t 20, 28 a n d 45°C l o w e r values w e r e o b t a i n e d .

 N I T R A T E R E D U C T I O N IN PLANT T I S S U E S 353

The experiments with leaf fragments of different sizes and with

leaf macerates (Table VII) have provided evidence that the nitrate-
reductase activity of the leaf fragments depends on the presence of
undamaged cells. Grinding of the ceils apparently inactivates the
enzyme system; in addition it may disorganize the electron-donor
system. Furthermore a loss of added nitrite was observed in leaf
macerates. The fact that added yeast extract, which is a source of
FAD, in some cases restored to a great extent the nitrate reduction
of ground leaf tissue, but in other cases restored it only partly or not
at all, may indicate that varying concentrations of reduced nucleo-
tides were, at least partly, responsible for the irregular results of
ground leaf tissues in the presence of yeast extract. That added FAD
and T P N H are able to restore nitrate-reductase activity of ground
leaf tissues m a y be concluded from the experiments of E v a n s and
N a s o n a and C a n d e l a et al. 2.
Illumination of cauliflower plants was found to have a depressing
effect on nitrate-reducing capacity only if the plants were previously
kept in the dark for a prolonged period of time. This is in agreement
with the results of similar experiments of C an d el aet oZ.s.
The effect of molybdenum on the nitrate-reducing capacity of
leaf fragments as observed in the present investigation m a y have
been due, in addition to its effect on nitrate reductase, to some
extent to the influence on malic-dehydrogenase activity, i.e. on
providing reduced nucleotides. This is concluded from the fact that
malic-dehydrogenase activity in some experiments was clearly
promoted by added molybdate. That the reduction of methylene
blue, which was used as a measure for malic-dehydrogenase activity,
was really brought about b y the activity of this enzyme was con-
cluded from the fact that omission of KCN from the reaction medium
retarded methylene-blue decolourization to a large extent.
The effect of molybdenum on malic dehydrogenase was less
consistent and far less pronounced than that on nitrate-reducing
capacity, however.
A further effect of molybdenum, which had been noticed earlier ~ 10
is its influence on catalase activity. This is an indirect effect,
however, and mainly depends, it is presumed, on newly synthesized
protein. This is concluded from the slow rise in catalase activity
upon addition of molybdate to molybdenum-deficient plants.
 354 E. G. MULDER, R. BOXMA AND W . L . VAN VEEN

SUMMARY

T h e effect of m o l y b d e n u m on t h e n i t r a t e - r e d u c i n g c a p a c i t y of p l a n t tissue
was s t u d i e d b y u s i n g coarsely c u t tissue f r a g m e n t s f r o m p l a n t s g r o w n in 3/[o~
deficient soil a n d dressed or i n f i l t r a t e d w i t h a m o l y b d a t e - c o n t a i n i n g s o l u t i o n
s h o r t l y before testing. T h e tissue f r a g m e n t s were s u s p e n d e d u n d e r a n a e r o b i c
c o n d i t i o n s in a n i t r a t e - c o n t a i n i n g b u f f e r s o l u t i o n w i t h m a l a t e s e r v i n g as a
h y d r o g e n donor. F o r m a t i o n of n i t r i t e was used as a m e a s u r e for t h e n i t r a t e -
r e d u c i n g power. V e r y low v a l u e s for n i t r a t e - r e d u c i n g c a p a c i t y were f o u n d in
m o l y b d e n u m - d e f i c i e n t tissues. As little as four h o u r s a f t e r t h e a p p l i c a t i o n of
m o l y b d e n u m , h o w e v e r , e n z y m e a c t i v i t y h a d r e a c h e d its m a x i m a l value.
Cauliflower, spinach, a n d t o m a t o p l a n t s , g r o w n w i t h i n a d e q u a t e a m o u n t s
of n i t r o g e n , s h o w e d also v e r y low n i t r a t e - r e d u c i n g activities. A d d i t i o n of
n i t r a t e to t h e s e p l a n t s , e i t h e r b y a p p l i c a t i o n t o t h e soil or b y i n f i l t r a t i n g i t
i n t o t h e leaves, r e s u l t e d in a r a p i d rise in n i t r a t e - r e d u c i n g c a p a c i t y . A m m o -
n i u m s u l p h a t e , a d d e d t o t h e soil, h a d some beneficial effect o n n i t r a t e re-
d u c t i o n in t h e leaves of cauliflower p l a n t s b u t in t h e case of i n f i l t r a t i o n n o
effect was observed.
E v i d e n c e was o b t a i n e d t h a t t h e n i t r a t e - r e d u c i n g a c t i v i t y of leaf f r a g m e n t s
d e p e n d s o n t h e p r e s e n c e of u n d a m a g e d cells. G r i n d i n g of t h e cells i n a c t i v a t e s
t h e e n z y m e s y s t e m ; in a d d i t i o n i t m a y d e s o r g a n i z e t h e e l e c t r o n - d o n o r
s y s t e m . F u r t h e r m o r e a loss of n i t r i t e m a y o c c u r in leaf m a c e r a t e s . A d d i t i o n of
y e a s t e x t r a c t , w h i c h is a source of f l a v i n a d e n i n e dinncleotide, a c o - e n z y m e of
n i t r a t e r e d u c t a s e , r e s t o r e d in some cases t h e n i t r a t e - r e d u c t a s e a c t i v i t i e s of leaf
m a c e r a t e s t o a large e x t e n t . I n o t h e r cases its effect was slight or negligible.
P l a n t s k e p t in t h e d a r k for a p r o l o n g e d p e r i o d of t i m e s h o w e d lower
n i t r a t e - r e d u c i n g capacities t h a n t h o s e k e p t u n d e r n o r m a l l i g h t conditions.
I n some e x p e r i m e n t s t h e m a l i e - d e h y d r o g e n a s e a c t i v i t y of m o l y b d e n u m ~
deficient p l a n t tissues was f o u n d to b e p r o m o t e d b y t h e a d d i t i o n of m o l y b -
date, T h i s effect was less c o n s i s t e n t a n d far less p r o n o u n c e d t h a n t h a t o n
nitrate-reducing capacity.
M o l y b d e n u m - d e f i c i e n t p l a n t tissues u s u a l l y h a v e a lower c a t a l a s e a c t i v i t y
t h a n p l a n t s supplied a d e q u a t e l y w i t h m o l y b d e n u m .
Received July 9, 1958

ACKNOWLEDGEMENT

T h e a u t h o r s are m u c h i n d e b t e d t o Dr. J. L. Harley, D e p a r t m e n t of B o t a n y ,

U n i v e r s i t y of Oxford, for r e a d i n g a n d i m p r o v i n g t h e t e x t of t h i s p a p e r .

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