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5108 K:IProj/Acc&Pres/CIRCIDateDue.hdd

ABSTRACT

THE INFLUENCE OF TEMPERATURE AND OTHER ENVIRONMENTAL
FACTORS ON NITRATE REDUCTASE ACTIVITY OF
AGROSTIS PALUSTRIS HUDS. AND
CYNODON DACTYLON L.

 

 

BY
John E. Kaufmann

The influence of temperature on nitrate reductase
activity was studied to determine if nitrate reductase is
associated with shoot growth inhibition at super- and sub—
0ptimal temperatures. The investigations were conducted in
controlled environment chambers with temperature variables
of 10°, 15°, 20°, 25°, 30°, 35°, and 40°C. Toronto bentgrass
and Tifgreen bermudagrass, the cool season and warm season
grass respectively, were chosen because of these distinctly
different temperature Optimums. The bermudagrass was
omitted from the 10°C temperature treatment, and the bent-
grass was omitted from the 40°C treatment because of
extremely adverse conditions for shoot growth.

The results indicated that nitrate reductase ac-
tivity did not decrease significantly under temperature

stress conditions until observed shoot growth inhibition

John E. Kaufmann

occurred at 35°C and up for bentgrass and 15°C and below

for bermudagrass. At high temperatures (35°C to 40°C)
samples of bentgrass exhibited an inhibition of growth and
nitrate reductase activity while bermudagrass was relatively
unaffected. This reduction of activity could be either an
inhibition of enzyme synthesis or inhibition of the activity
per g3. It was found that the activity p§£_§g of the bent-
grass enzyme preparation was also inhibited by these same
temperatures, suggesting that nitrate reductase inhibition
may be a factor causing high temperature growth inhibition
in this species.

The investigation also indicated that when bentgrass
is grown at 15°C and bermudagrass at 30°C, the nitrate
reductase activity was higher in the leaf blade tissue than
in stem tissue, was higher following application of a
nitrate nutrient solution, increased with increasing light

intensity, and increased with length of light exposure.

THE INFLUENCE OF TEMPERATURE AND OTHER ENVIRONMENTAL
FACTORS ON NITRATE REDUCTASE ACTIVITY OF
AGROSTIS PALUSTRIS HUDS. AND

CYNODON DACTYLON L.

BY

John E. Kaufmann

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Crop and Soil Sciences

1970

 

)r.

ACKNOWLEDGMENTS

The author wishes to express his sincere apprecia—
tion to Dr. J. B. Beard for his guidance and encouragement
throughout this study and for his constructive criticism in
the preparation of this manuscript.

Grateful appreciation is expressed to Dr. D. Penner
for his many helpful suggestions and critical review of the
manuscript during Dr. Beard's absence.

Appreciation is also expressed to Dr. P. Rieke and
Dr. S. N. Stevenson for serving on the guidance committee.

Special thanks is given to my fiancee, Jean Hamilton,
for her assistance in typing and reviewing the original

manuscript.

ii

LIST OF TABL
LIST OF FIGU

INTRODUCTION

TABLE OF CONTENTS

ES 0 O O O O O O O I O O C O C O O 0
RES 0 O O O O O O O O O O O O O O O O

LITEMTURE REVIEW 0 O O I O O O O O O O O O O O O

Source and
Pathway of
Effects of
Properties
Cofactors

Effect of

Physiologi
Temperatur

MATERIALS AN

Establishm
Growth Con
Preparatio
Assay Proc

RESULTS AND
Effect of
Effect of
Effect of
Effect of
‘Effect of

CONCLUSIONS

BIBLIOGRAPHY

APPENDIX .

Assimilation of Nitrogen . . . . . .
Nitrate Reduction . . . . . . . . .
Inhibitors and Stimulators . . . . .
and Functions of the Enzyme and
Hereditary Factors . . . . . . . . .
cal Responses of Turfgrasses to

e O O O O O O O O O O O O C C O I C O

D METHODS O O O O O O O O I I O O O O

ent Procedures . . . . . . . . . . .
ditions for Temperature Studies . . .
n of Nitrate Reductase . . . . . . .
edure . . . . . . . . . . . . . . . .

DISCUSSION . . . . . . . . . . . . .

Tissue Sampling on Nitrate Reductase
Nutrients on Nitrate Reductase .
Light on Nitrate Reductase . . .
Temperature on Nitrate Reduction
Temperature on Enzyme Activity .

iii

Page

iv

0% Chub-w

KO

14

14
16

18
21
21
23
24
26
29
33
35

41

- fim'nfl

 

LIST OF TABLES

Table Page

1. The effect of tissue sampling on nitrate
reductase activity . . . . . . . . . . . . . . 22

2. The effect of nutrient application on nitrate
reductase activity . . . . . . . . . . . . . . 23

3. The effect of light intensity on nitrate
reductase activity . . . . . . . . . . . . . . 25

4. The effect of length of light exposure on
nitrate reductase activity . . . . . . . . . . 25

iv

LIST OF FIGURES

Figure
1. The effect of temperature
of nitrate reductase of
and bermudagrass . . .

2. The effect of temperature
of nitrate reductase of
and bermudagrass . . .

Page
on the induction
creeping bentgrass
O O O O O O O O O O O 27
on the activity
creeping bentgrass
O O O O O O O O O O O 30

INTRODUCTION

Toronto creeping bentgrass, Agrostis palustris Huds.,

 

is one of the most widely used, vegetatively established
creeping bentgrasses on greens in the northern cool-humid
climatic regions of the United States. It is commonly

subject to growth inhibition at high temperatures, espe—
cially under the cultural conditions practiced on greens.

Tifgreen bermudagrass, Cynodon dactylon L., is the

 

counterpart of bentgrass for greens in the warm-humid cli-
matic regions. Bermudagrass exhibits growth inhibition
more commonly at suboptimal temperatures.

The temperatures at which growth inhibition occurs
are common in the regions where each grass is cultivated.
Under prolonged periods of temperature stress high tempera—
ture results in bentgrass kill, and low temperature in
bermudagrass dormancy.

Nitrate reductase is an important enzyme in the
pathway which provides nitrogen to the plant in a form
which is readily available for the synthesis of essential
nitrogen compounds such as amino acids. It is considered
by Hageman (20, 21) and others to be a limiting factor in

plant growth.

The objectives of this investigation were to deter-
mine the effect of certain major environmental factors on
nitrate reductase, and to study whether the influence of
temperature on nitrate reductase activity of these species

is related to growth inhibition under temperature stress.

LITERATURE REVIEW

In some of the early studies concerning the occur-
rence of nitrate (N03) in plants Berthelot (as reported by
Anderson (2)) found that the N03 content may vary greatly
during the life cycle of the plant. Also, NO3 levels were
localized in various parts of the plant, and were dependent
on the availability of NO3 in the soil. Anderson further
reports the studies of (a) Kastle and Elove and (b) Haas
and Hill, as indicating a mechanism for nitrate reduction
in potato sap and milk respectively. Conclusions from this

work indicate the probability that an oxidizable substance

is involved rather than an enzyme system.

Source and Assimilation of Nitrogen

NO3 assimilation has been studied through measure-
ment of the loss of NO3 from nutrient solutions. The
assimilation of NO3 was absent in darkness and increased
with increasing light intensity (8). N03 assimilation
paralleled CO2 assimilation and at the same time utilized
no carbohydrates. Ammonium (NH4) assimilation required
carbohydrates at all times and was assimilated both in the

light and dark. However, addition of glucose stimulated

nitrate assimilation in Chlorella vulgaris (54).

 

3

5

Through the use of nitrogen (1 N), assimilation

rates of NO and NH were measured. Light and dark did not

3 4
influence on the total amount of 15N assimilated (12).
Tomato plants fed 15NH4.had a high concentration of 15N in

the roots while plants fed 15NO accumulated 15N in the

3

leaves (31). Studies with Chlorella vulgaris show that NH4

is assimilated four times as fast as NO3 in nitrogen-starved

 

cells (55), and that NO3 assimilation is inhibited during
NH4 assimilation (56). Morton (34) reports that NH4 in-
hibits the reduction of nitrate in fungi because of direct
ammonia (NH3) assimilation.

Fertilization with N03 has been shown superior to

NH4 in supporting better shoot growth and rhizome develop-

ment in Poa pratensis L. (23), and has been shown to be

 

effective over a wider range of soil pH and temperature

(11). Recent work on Lolium multiflorum Lam. indicated

 

 

that at cool temperatures high rates of NO3 were more
effective in promoting growth than high rates of NH4. At
high temperatures, NO3 was again more effective than NH4

at both high and low levels of nutrition (51).

Pathway of Nitrate Reduction

Nitrate assimilation involves a pathway of nitrate
reduction to provide the plant with the nitrogen form
necessary for protein synthesis. Many pathways from N03

to NH3 have been prOposed and reviewed (9, 27, 31, 35, 59,

60). However, Burstrom (9) lists the possible intermediates

of nitrate reduction:

H NO3 Nitric acid

H NO2 Nitrous acid

(H NO)2 Hyponitrous acid
H NO H2 Hydroxylamine

H N H2 Ammonia

Of these five intermediates, hyponitrous acid has never
been shown to exist in the plant. The presence of nitric
acid, nitrous acid,and ammonia in the plant is easily
detected. Hydroxylamine has been shown to exist in the
leaves of higher plants following the addition of N03.

According to Webster (60), Zucker and Nason have
actually isolated a hydroxylamine reductase enzyme in
neurospora. Betts and Hewitt (6) indicate the chlorOplasts
of higher plants are the sites for photochemical reduction
of nitrite and hydroxylamine. They suggest that both com-
pounds are reduced by the same or closely related enzymes,
indicating that hydroxylamine is not a true intermediate in
the pathway.

This pathway of nitrate reduction is a series of
endothermic reactions (8, 9). All steps which have been
shown to occur require a reductase enzyme and a series of
electron donors referred to as reaction cofactors. The

following discussion of the enzyme and cofactors will be

to N0 .

limited to the first step of reduction, namely N03 2

Effects of Inhibitors and
Stimulators

 

 

In Chlorella vulgaris NO assimilation appears to

 

3
be inhibited during NH4 assimilation, perhaps due to the

inhibition of the nitrate reductase enzyme (52). Further
study indicates that carbamyl phosphate when in alkaline
solution, produces a cyanate which inhibits nitrate reduc-
tion. Carbamyl phosphate may be the primary product of
NH4 assimilation (33).

The presence of a metal constituent in the nitrate
reductase system is shown by the effect of cyanate, a known
metal binding inhibitor (16, 47). The presence of sulfhydryl
groups on the enzyme is substantiated by the inhibitory
effect of p-chloromercuribenzoate. The inhibition is easily
reversed through the addition of a known sulfhydryl-group
containing compound such as cysteine (16, 47).

Carbon monoxide (CO) inhibits nitrate reductase in
the dark only, and is reversed by light. This suggests
that a specific cytochrome of photosynthesis is utilized in
the electron transport system of nitrate reductase.
Benzylviologen also reverses the inhibitory effect of CO
apparently through by—passing the cytochrome (47). A more
recent investigation of a purified nitrate reductase prep-
aration shows benzylviologen as a capable substitute for

the natural flavin cofactors (41).

Studies of the effect of 2,4-D on nitrate reductase
activity shows an increase in §§a_m3y§ L. and a decrease in
cucumbers (4). It was speculated that 2,4-D causes an
intramolecular change in the protein sulfhydryl groups.

Simazine has been shown to stimulate the assimila-

tion of NO however, the study was conducted at sub-

3!
Optimal conditions of low NO3 and low temperature levels
(57).

Most of the amino acids present at specific concen—
trations not uncommon to the cell, have an effect on nitrate
reductase activity (18). Of two categories described,

repressors and derepressors, methionine and alanine are

repressors while arginine and lysine are derepressors.

Properties and Functions of the
Enzyme and Cofactors

 

 

The nitrate reductase enzyme is thought to be a
sulfhydryl-metallo-flavoprotein (16, 17, 35, 47). The
sulfhydryl group is considered to be the site on the enzyme
which binds the unreduced cofactor or electron donor. Many
electron donors are shown to be involved in nitrate reduc-
tion. The most widely recognized being the flavin require-
ment of either FMN or FAD and either NAD, or NADP, or both.
In some studies the enzyme system was specific for NAD

(14, 46, 50), while another was NADP specific (28).

The metal constituents may vary with plant species.
Molybdenum, a minor element, has been shown to be a cofactor
for nitrate reduction (37). It has been found to serve as
an electron donor in bacteria (17), fungi (35, 39, 40), and
in higher plants (24, 27) with specific studies in tomatoes
(58) and soybeans (15, 38).

The role of phosphate in nitrate reduction is more
indirect than molybdenum. Nicholas and Nason (38) indicate
that nitrate reductase in soybeans has no requirement for
inorganic phosphate, while Spencer (50) shows a need for
phosphate in a nitrate reductase system of germinating
wheat embryos. Further study suggests that phosphate
either binds molybdenum into a phosphomolybdenum complex
(28), or serves in binding molybdenum to an apoenzyme of
nitrate reductase (27).

The role of another metal is considered part of the
system through the fact that light influences nitrate
reduction. The electron transport system of photosynthesis
requires iron and in this sense has been reported as a
requirement for nitrate reductase (17). Another investiga-
tion, reviewed by Nason and Takahashi (36) found that the
cOpper ion may substitute for iron. Extensive work has
been done to formulate the series of cofactors responsible
for providing electrons for the nitrate reduction system.
Extreme variation between species of proposed pathways

makes it impossible to record a general form. Most pathways

include (a) grana of the chloroplast (29, 16) or chlorophyll
(45), (b) one of the many cytochromes (17, 28, 36, 37, 47),
(c) the flavins, (d) NAD or NADP, and (e) the heavy metals

previously discussed.

Effect of Hereditary Factors

 

Nitrate reductase activity is known to vary among
the genetic lines in corn (ESE.TEX§.L°) (61). Hybrid
crosses from an inbred line of high nitrate reductase ac-
tivity and an inbred line of low activity proved to exhibit
enzyme activity intermediate between the two parental in-
breds (22, 48). With these hereditary factors in mind, a
hybrid could be developed with a nitrate reduction system
which would compliment the other improved factors of the

hybrid.

Physiological Responses of Turfgrasses
to Temperature

 

 

Cool and warm season grasses exhibit different
growth responses when placed under various temperature
regimes. Early observations on colonial bentgrass (Agrostis
tenuis Sibth.) indicate that at a temperature of 90°F
(32.2°C) as compared to 60°F (15.5°C) the seeds germinated
more rapidly, the plants grew more rapidly but lacked vigor,
and 4 weeks later chlorosis occurred under the 90°F (32.3°C)

treatment. At 80°F (26.6°C) the topgrowth tended toward a

10

horizontal position, and the roots were white, fleshy, and
unbranched (52).

Kentucky bluegrass is described as producing suc-
culent, bushy shoot-growth at 15°C while the shoot growth
at 35°C is less succulent, shorter, and the shoot density
is severely thinned (11). After a 40 day exposure to day-
night temperatures of 90—80°F (32.2-26.6°C) respectively,
the shoot growth of perennial ryegrass (Lolium perenne L.)
was severely stunted, the leaves were spindly and dark green
in color, and the roots were fibrous and discolored (53).

Early work by Brown (7) showed no reduction in shoot
growth as temperatures were increased from 80°F (26.6°C) to
100°F (37.7°C), whereas a reduction was observed in cool
season grasses. The curve of temperature and leaf matter
production of bermudagrass indicates that growth steadily
increases from 40°F (4.4°C) to 100°F (37.7°C). Maximum
shoot growth occurred at 100°F (37.7°C). A growth curve
for perennial ryegrass indicates that growth peaked at 65°F
(12.7°C) with a slight reduction in growth at 45°F (7.2°C)
and total inhibition of growth at 95°F (35°C) (32). In a
recent and comprehensive study of temperature effects on
TOronto creeping bentgrass, Duff (13) provides a growth
curve reported as dry matter production per week. The
temperatures were day-night regimes of 20-10°, 25-15°, 30-
20°, 35-25°, and 40-30°C. A gradual decrease in shoot

growth was noted from 20—10°C to 30—20°C. A sharp reduction

11

occurred between 30-20°C and 35-25°C, and at 40-30°C growth

cessation occurred. Duff states that increasing temperature
increased the percentage dry weight of the tissue produced,

and decreased the leaf width.

Another factor is interaction of nutrition and
temperature levels. Harrison (23) concludes that the high
levels of nitrogen are undesirable at high temperatures.
Pellett and Roberts (42) state that turf grown on low levels
of nitrogen is more heat tolerant than turf grown on high
nitrogen levels. They propose an apparent upset of growth-
differentiation balance where at high nitrogen levels, the
carbohydrates are utilized for growth and are not available
for differentiation.

In the case of the cool season grasses such as
Toronto creeping bentgrass, temperatures of 80°F tend to
exert an adverse effect on carbohydrate reserves (19).
Sullivan and Sprague (53) state that high temperatures
cause a rapid dissipation of reserve carbohydrates and a
possible toxic formation of nitrogen compounds. Hewitt
and Curtis (25) contend that high temperatures increase
respirational and translocational losses of carbohydrates.
At extremely high temperatures respiration of transportable
materials occurs.

Recent work, however, refutes the theory that carbo-
hydrate 1033 causes growth stoppage at high temperatures.

While the loss of carbohydrate reserves from the root system

12

may take place, Duff (13) clearly states that leaf tissue
of Toronto creeping bentgrass actually exhibits an approx-
imate 50% increase of both water soluble and 85% ethanol
soluble carbohydrates when grown at super-Optimal tempera-
tures. Photosynthesis and respiration both increased as
temperatures were increased. The chlorOphyll content
drOpped severely at these same temperatures although the
leaves were visibly darker green.

In further study of high temperature stress, Petinov
and Molotkovskii (44) propose four mechanisms: (a) When
respiration is inhibited, the tissue exhibits less tolerance
to heat; (b) Proteolysis is intensified and ammonia accum-
ulates; (c) Amino acid synthesis ceases and abnormal amino
acid metabolism occurs; and (d) ReSpiration is concluded to
be the source of active metabolites and energy for resyn-
thesis of proteins destroyed by high temperature. In a
second study, Petinov and Molotkovskii (43) describe the
organic acids as the active metabolites responsible for
resynthesis of proteins. They state that plants with high
temperature resistance form organic acids during the respi—
ratory process which combine with ammonia (reducing ammonia
tOxicity) to form amides and eventually new proteins. They
further state that the accumulation of ammonia is the
meChanism which triggers the defense reactions forming the

organic acids.

13

The quantities of various nitrogen compounds in
leaf tissue of bentgrass and bermudagrass, over a range of
four constant temperatures (50°, 60°, 70°, and 80°F), have
been reported by Beard (3). Total nitrogen content in-
creased in both species with increasing temperature. Non—
protein nitrogen increased with increasing temperature in
bentgrass but decreased with increasing temperature in
bermudagrass. The free ammonia level increased from 50°
to 70°F and decreased at 80°F in both species. The amide
level in bermudagrass decreased with increasing temperature.
A continuing reduction in amide levels is noted with in-
creasing temperature in bentgrass. Bermudagrass, when
grown at 80°F, appears to have a mechanism to reverse this

downward trend.

MATERIALS AND METHODS

Establishment Procedures

 

Mature sod of Toronto creeping bentgrass was ob-
tained from the Michigan State University experimental
turfgrass field laboratory where it had been maintained at
0.25 inch cutting height. Circular plugs (3.5 inch diam-
eter by 3 inch depth) were removed with a cup cutter and
taken to the greenhouse for preparation.

The Tifgreen bermudagrass, also maintained at 0.25
inch cutting height, was obtained from the Plantation Field
Laboratory, Ft. Lauderdale, Florida, and was mailed in sod
strips (2' by 2') to Michigan State University. The sod
pieces were plugged and prepared for establishment.

A soil mix of 50% sand and 50% sandy loam topsoil
was poured into waxed cups (3.5 inch diameter by 6 inch
depth) to a height 1.5 inch from the rim. The sod plug,
which had been trimmed to a 1 inch soil depth, was firmly
pressed on top of the soil mix. This procedure allowed
0.5 inch of shoot growth to reach the cup rim.

Thirty six cups of each species were prepared in
this manner. Drainage holes were punched in the bottom of

the cups which were placed in the greenhouse under an

14

15

automatic watering system for 2 weeks. Following the
establishment period the plugs were placed in controlled
environment chambers and watered twice daily with a full
nutrient solution.

This nutrient solution was similar to Hoaglands
(26) solution, with modification of N, P, and K to a 4:1:2
ratio respectively. Minor elements were added according to
Hoagland. Two-thirds of the nitrogen was applied as NO3
and one-third as NH4. The concentration of the nutrient
solution was adjusted to apply an approximate rate of 4 lbs.
N/1000 sq. ft./month. The bentgrass was placed in a 15°C
chamber, and the bermudagrass was placed in a 30°C chamber.
The temperatures chosen corresponded to the optimum growth
rate of each species. Light intensity was adjusted at 2,000
foot candles, and the daylength was set at 16 hours, with
day-night temperatures held constant. Dyrene, a turf
fungicide, was applied every other week for disease pre-
vention, and an occasional dusting with malathion was
necessary for insect control.

The harvest consisted of a daily clipping of the
grass to a 0.5 inch height in each cup. The sample was
composed primarily of leaf tissue with very little stem
tissue collected. The cup was held at a 45° angle and a
scissors was used in such a motion as to cause the clippings
to fall on a piece of paper. The clippings were then

immediately transferred to a 4 ounce bottle of ice cold

16

distilled water. The bottle containing the sample was

immediately placed in an ice bucket and kept near 0°C.

Growth Conditions for Temperature
Studies

 

In the experiment designed to determine the induc-
tion or availability of the nitrate reductase enzyme at
various temperature levels, two growth chambers were used
with environmental factors carefully controlled in each
chamber.

The two chambers were first adjusted to 30°C and
20°C. Eighteen plugs of each species were placed in each
chamber. The following week the chambers were adjusted to
35°C and 15°C. Six plugs of bentgrass were removed from
15°C and placed in 35°C, and six plugs of bermudagrass were
transferred from 35°C to 15°C in order to provide the nec-
essary one gram (fresh weight) tissue per day as the grasses
approached temperature stress. The third temperature levels
were 10°C and 40°C. At these temperatures all the bentgrass
was placed in the Cool chamber and all the bermudagrass was
placed in the warm chamber. The turf was allowed to adjust
to each temperature level for one week and harvests, con-
sisting of one composite sample from all cups of each
species, were taken on the 5th, 6th, and 7th day of each

week.

17

The final temperature was 25°C. All of the turf
was placed in the same chamber. Twelve cups of corn seed-
lings grown with 4 seedlings per cup, were placed in the
chamber and watered with the same nutrient solution. At
the three leaf stage, leaf tissue was harvested and analyzed.
The corn variety (Hy2 x 0H7), was used to provide a refer-
ence point to the work done by Hageman (20).

In the experiment designed to determine the actual
activity of the nitrate reductase enzyme system over a range
of temperatures, the turf was maintained at 25°C for 2 weeks
prior to harvesting. One large sample was taken of each
species. The four temperatures at which the enzyme reaction

was determined, were 10°, 20°, 30°, and 40°C.

Preparation of Nitrate Reductase

 

In the laboratory the entire sample was removed
from the bottle with the aid of a hooked glass rod. The
sample was blotted dry in paper toweling, cut into small
pieces, and weighed. Exactly one gram was removed and
placed in a blending cup.

The extraction proceeded according to the methods
Of Hageman and Flesher (20) with few modifications. The
extraction medium, consisting of 0.1 M Tris buffer, 0.01 M
cysteine, and 0.0003 M EDTA, was added to the sample in the
ratio of 15 ml per gram of tissue. Cysteine was added to

the medium to protect the sulfhydryl-groups of the enzyme.

18

EDTA, a metal cheleting agent was used to bind excessive
amounts of heavy metal ions. The pH was adjusted to 7.2
with HCl prior to each extraction.

A model "45" Virtis homogenizer was used to grind
the sample; one minute at slow speed to cut the tissue, and
two minutes at maximum speed to blend the sample. Due to
excellent blending it was not necessary to press the homo-
genate through cheese cloth before centrifuging for 20
minutes at 20,000 x g. The supernatant was carefully
decanted into test tubes. The temperature of the enzyme
preparation was maintained at 0 to 3°C throughout the

entire extraction procedure.

Assay Procedure

 

The assay procedure was based on the method of Evans
and Nason (l6), and was similar to the procedure of Hageman
and Flesher (20). The assay mixture included 1.5 ml of
0.1 M potassium phosphate buffer (pH 7.0), 0.5 ml of 0.1 M

3

KNO , 1.0 m1 of 1.36 x 10' M NADH and 1.0 ml of the

2:
nitrate reductase enzyme preparation. Potassium phosphate
and KNO3 were pipetted into test tubes and allowed to adjust
to the incubation temperature of 28°C. The reaction was
started with the addition of NADHZ, followed immediately by
the enzyme extract. Exactly 12 minutes later the reaction

was stopped by adding 2.0 ml of 1% w/v sulfanilamide in

1.5 N HCl. N-(l-napthyl) ethylene-diamine-dihydrochloride

19

reagent (2.0 ml of 0.0228% w/v) was immediately added and
the entire volume of 8 ml was thoroughly mixed.

Following the procedure reported by Snell and Snell
(49) the color was allowed to develop for 10 minutes. The
absorbancy was determined by reading each sample against
its own blank (complete except for NADH) in a Beckman DU
spectrOphotometer at 540 mu. Samples were drawn from the.
bottom of the tube and analyzed quickly and accurately with
the aid of a flow-through cell.

The samples were completely analyzed within two
hours of harvest. Enzyme extracts that were heated for
five minutes in boiling water were inactive. The activity
of-the nitrate reductase was expressed as micromolar po-
tassium nitrite (uM KNOZ) formed per gram fresh weight per
hour.

The final expressed activity was the result of the
average absorbancy of two blanks subtracted from the average
of three determinations for each sample, and transposed,
through the use of a predetermined standard curve, into
uM KNOZ. Replication was achieved by repeating the exper—
iment on the following day again using fresh plant material.
I In measuring the temperature stability of the
enzyme, the above procedure was slightly modified. The
incubation temperature normally at 28°C was adjusted to

10°, 20°, 30°, and 40°C, with the aid of 4 water baths.

20

Also, following the final step, the reaction mixtures were
immediately removed from the water baths to avoid possible

differentiation in breakdown of the red color compound.

RESULTS AND DISCUSSION

In order to determine Optimum conditions for enzyme
activity, it was necessary to study various factors which
have been reported to have an effect on nitrate reductase
activity. Each environmental factor was controlled as
described in the plant establishment procedures unless it

was being examined in the specific study.

Effect 9f Tissue Sampling on
Nitrate Reductase

 

 

Six plugs of bermudagrass were allowed to grow to a
4 inch height, while another six were maintained at a 0.5
inch height. The grass was allowed to adjust to these
conditions for one week. At that time 0.5 inch of tissue
was harvested from the shorter grass and 2 inches was har-
vested from the longer grass. In the latter, the 3 to 4
inch increment and the 2 to 3 inch increment were harvested
as separate samples. The 2 to 3 inch increment consisted
of a stemmy tissue, being about 50% leaf sheath.‘ The sam—
ples were analyzed for nitrate reductase activity. The
greatest activity was found in the grass maintained at the
0.5 inch height (Table 1). Also the leaf blade tissue
exhibits more nitrate reductase activity than tissue con-

taining 50% leaf blade and 50% leaf sheath tissue.
21

22

Table 1.--The effect of tissue sampling on nitrate reductase
activity.

Bermudagrass 30°C

 

 

 

Tissue Length Activity uM KNOz/g.F.wt./Hr.
I II Average
1" to 0.5" 48.0 45.0 46.5
4" to 3" 11.0 7.0 9.0
3" to 2" 6.0 5.0 5.5

 

According to other studies, nitrate reduction takes
place primarily in the leaf tissue. In working with cauli-
flower, Candella, EE;.E£° (10) reported greater nitrate
reduction in the leaves than in the petioles or roots, and
that mature leaves yielded extracts with maximum activity.
Wallace and Pate (59) report in their work with peas that
enzyme induction occurs most readily in actively growing
tissues; hence the newly expanded leaf exhibited the highest
activity. Hageman and Flesher (20), using corn seedlings
state that the activity of nitrate reductase was 80% lower
in root extracts as compared to extracts of shoots and
leaves.

Since each cup was filled with soil to within 0.5
inch of the rim, accurate and reproducible harvest methods
allowed the grass to grow 0.5 inch which was then clipped
back to the rim of the cup. At optimum temperatures for

growth, 0.5 inch of-shoot growth was attained daily.

23

Effect of Nutrients on Nitrate
Reductase

 

 

Twelve plugs of bentgrass were clipped daily and
watered with tap water for one week. At the end of the
week the plugs were harvested and the nutrient solution
was immediately applied. A complete nutrient program was
maintained and harvests were again made at 24 and 48 hours
following the initial harvest. The samples were analyzed
for nitrate reductase activity. Within 24 hours after
nutrient application, the enzyme activity was induced to a
maximum rate (Table 2). A slight drop in activity was
noted at 48 hours.

Table 2.--The effect of nutrient application on nitrate
reductase activity.

Bentgrass 15°C

 

 

 

Nutrient Application Activity uM KNOz/g.F.wt./Hr.
I II Average

0 Hrs. 0.0 0.0 0.0

24 Hrs. 85.0 72.0 78.5

48 Hrs. 72.0 75.0 73.5

 

Related studies show that N03 is the principle form
of nitrogen absorbed by higher plants and induces higher
nitrate reductase activity than NH4 (10). Low fertility
levels result in low activity of nitrate reductase (30).

The nitrate reductase activity in corn seedlings increased

24

with increasing concentrations of N03 (20). Beevers, eE;_al,
(5) reported that the induction of nitrate reductase is
proportional to, and dependent on the amount of NO3 in the
tissue. Relative amounts of N02 or NH3 had no effect. The

availability of NH did not inhibit the induction of the

4
nitrate reductase enzyme.

The time of application of nutrients had an effect
on enzyme activity. However, this could not be separated
from a moisture stress effect since the plugs received no
water other than the full nutrient solution. Nutrients

were applied twice daily to overcome moisture stress at

high temperatures.

Effect of Light on Nitrate
Reductase

 

 

Twelve cups of bermudagrass were placed in a 30°C
chamber at a light intensity of 2,000 foot candles. After
a one week establishment period, six were moved to a 30°C
chamber with new bulbs producing 2,400 foot candles. After
48 hours, each set of six cups was harvested and analyzed
for nitrate reductase activity. An increase in light
intensity increases the activity of the enzyme (Table 3).

The time of day of harvest also had an effect on_
nitrate reductase activity. Six plugs of bentgrass were
harvested at 8:00 a.m. (after 3 hours of daylight), and six

plugs were harvested at 8:00 p.m. (after 15 hours of

25

daylight). Each of the samples were analyzed for enzyme
activity (Table 4). The higher activity at 8:00 p.m.
indicated that a period of light exposure was necessary
to obtain maximum activity.

Table 3.--The effect of light intensity on nitrate reductase
activity.

Bermudagrass 30°C

 

 

 

Light Intensity Activity uM KNOz/g.F.wt./Hr.
I II Average
2,000 F.C. 42.0 45.0 43.5
2,400 F.C. 49.0 53.0 51.0

 

Table 4.-—The effect of the length of light exposure on
nitrate reductase activity.

Bentgrass 15°C

 

 

 

Length of Activity uM KNOz/g.F.wt./Hr.
Light Exposure I II Average
3 Hours 51.0 50.0 50.5
15 Hours 65.0 78.0 71.5

 

Light is known to be a very important, and perhaps
essential factor in nitrate reduction through the capability
of the photosynthetic process to donate the necessary
electrons for reduction of N03. Hageman and Flesher (20)

have extensively studied the various effects of light;

26

including a reduction in enzyme activity due to partial
shading and total darkness, and a subsequent increase upon
light restoration. Diurnal variation was such that a
sampling made at dawn had approximately 50% the activity
level of a sample taken at midday. The enzyme activity of
plants under artificial shade also showed a reduction when
sampled in early morning. However, the difference between
shade and full sun was much greater in the midday sample
(21) .

It is impossible to maintain the growth chambers at
2,400 foot candles for any length of time, so the light
intensity was maintained at 2,000 foot candles and was
checked bi-weekly with a light meter. All harvests were
carried out at 12:00 noon (after 7 hours daylight), with

analysis of the enzyme following immediately.

Effect of Temperature on Nitrate
Reduction

 

 

In determining the effect of temperature on nitrate
reductase activities one species with, and one without high
temperature resistance was used. The temperature effect on
the apparent availability and actual activity of the nitrate
reductaSe enzyme was studied.

Nitrate reductase appeared to be induced at 20°C in
both species, with warmer temperatures reducing the activity

that could be isolated (Fig. 1). Following the peak of the

27

Figure l.--The effect of temperature on induction of nitrate
reductase of creeping bentgrass and bermudagrass.

 

 

80 I?
O
60 p
0‘.
- r
L: \.
III
\. \\\\\
g 40 . O
. O O
”S \a
\.
ON
2 L
M
E
:1.
20 ’
O
L
Bentgrass .———O
Bermudagrass o——-o
0 A A A L A L L I
10 20 30 40

TEMPERATURE ° C

*Nitrate reductase activity of corn (cultivar
Hy2 x 0H7) to provide a point of reference to work done by
Hageman (20).

28

curve at 20°C, the activity of the enzyme drOpped signif-
icantly, but seemed to be maintained at an adequate level
of growth for both species through 30°C.

The activity level of the corn seedlings grown at
25°C was comparable to that of both bentgrass and bermuda-
grass. Variations in the technique, as compared to Hageman
(20) are probably responsible for the difference in the
reported levels of EM KNO2 per gram fresh weight per hour.

The level of nitrate reductase in bermudagrass
seemed adequate for growth at 15°C. However the turf
exhibited little growth at this temperature, indicating
that under these conditions nitrate reductase activity was
not limiting growth.

At high temperatures, bermudagrass exhibited only
slight reductions of enzyme activity from 25° to 45°C.
These levels of activity found for 25°, 30°, 35° and 40°C,
did not greatly differ from the activity noted at 15°C.

The nitrate reductase activity of bentgrass was
greater at 10°C than at.25°C or higher temperatures, corre-
lating with the cool season growth pattern of this species.
The peak of the curve was noted at 20°C. High temperature
Vstress of this species, noted at 35°C, sharply reduced both
the activity of the nitrate reductase enzyme (Fig. l) and
growth (13).

At-temperatures of 35°C and higher, the amount of

nitrate reductase activity that could be isolated from the

29

bentgrass was much lower than for the bermudagrass. Either
there was less enzyme present in the bentgrass at high tem-
peratures, or the enzyme was present but inactive. Subse-

quent studies were initiated to study the latter possibility.

Effect of Temperature on Enzyme
Activity

 

A differential effect of temperature on the activity
225 §g_of nitrate reductase was observed between the two
species (Fig. 2). From 10° to 30°C both species exhibited
normal enzyme-temperature curves of increased temperature
yielding increased activity. In bermudagrass the activity
at 40°C remains at the same level found at 30°C, while in
bentgrass the activity at 40°C drops to approximately the
level found at 10°C.

Temperature is an important factor influencing both
nitrate reductase induction and activity. The induction of
nitrate reductase is temperature dependent, however, some
of the effect may be due to the temperature effect on
increased uptake of N03 (5). Afridi and Hewitt (1) report
that when N03 is added at various temperatures, increasing
enzyme activity was noted with increasing temperature up to
a critical temperature. Mattas and Pauli (30) point out
that it is difficult to distinguish between temperature and
moisture stress. They examined this temperature-moisture

stress combination and found that a reduction in nitrate

30

Figure 2.--The effect of temperature on the activity of
nitrate reductase of creeping bentgrass and

40

30

N
0

uM KNOZ/gF.wt./Hr.

I—'
O

 

 

 

 

bermudagrass.
O'-\
to
/.
P o/
/.
I. .
H O/ .
O
I
Bentgrass O O
Bermudagrass o' O
— ‘ ‘ ‘ ‘ ‘ ‘ H
10 20 30 40
TEMPERATURE °C

31

reductase activity occurred before the stress was measurable
through relative turgidity.

The temperature for inactivation of bentgrass
growth (13), and observed nitrate reduction (Figs. 1 & 2)
is between 30°C and 40°C. In this same temperature range,»
bermudagrass exhibits adequate growth and nitrate reduction.
The difference between these two species may be described
as the difference in adaptation to environment through
evolutionary processes. But this approach does not answer
the question of why bentgrass lacks heat resistance. Sev-
eral prOposals have previously been suggested to answer
this question.

It was first thought that increased respiration at
high temperatures depleted the carbohydrates in the plant.
Duff (13) clearly shows that this is not the case in bent-
grass. Ammonia toxicity was also proposed as a mechanism
of high temperature growth stOppage in the plant. As an
end product of nitrate reduction, ammonia could become
concentrated in leaf tissue causing the lack of heat tol-
erance. However, the high temperature inactivation of
nitrate reductase in bentgrass leaf tissue shown in Fig. 2‘
‘seems to indicate that if a toxic level of ammonia exists
at high temperatures, it is not produced by the pathway of
nitrate reduction. High temperatures can also increase
proteolysis, which may increase NH3 levels. If, in this

breakdown of protein, NH3 is not utilized through protein

32

resynthesis, toxic levels of NH could eventually occur and

3
possibly inhibit nitrate reductase synthesis or activity.
Figure 1 indicates that the enzyme activity in
bentgrass is reduced when the sample is harvested under
high temperature conditions. This reduction is due to
either high temperature inhibition of the enzyme synthesis
or high temperature inactivation of the enzyme. While both
of these activity reductions may occur in bentgrass at high
temperature, Figure 2 indicates that high temperature

inactivation of the enzyme is responsible for at least part

of the reduced activity found in Figure l.

CONCLUSIONS

As reported in studies using other species, the nitrate
reductase activity of Toronto creeping bentgrass and
Tifgreen bermudagrass is found to reSpond to these
factors.

a. The activity of nitrate reductase is higher in the
leaf blade tissue than in stem tissue.

b. The activity of nitrate reductase increases upon
the addition of a nitrate nutrient solution.

c. The activity of the enzyme increases with increasing
light intensity and increasing periods of light
exposure.

Temperature has a differential effect on nitrate reduc-

tase according to species. The enzyme system of both

Species appears to be induced at 20°C but at high

temperatures induction may be inhibited in bentgrass

while it remains adequate in bermudagrass.

Temperature also has a differential effect on in—
activation of the enzyme between the two Species.
Increasing temperature increases the activity of the
enzyme through 30°C. At 40°C the enzyme of bentgrass
is inactivated while no inactivation is found in
bermudagrass.

33

34

If nitrate reductase is a limiting factor of growth,
the inactivation of the enzyme per §g_of bentgrass grown at
high temperatures may explain the high temperature growth

inhibition of this species.

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AP PEND IX

 

Appendix Table 1.--The nitrate reductase activity of
bentgrass expressed as uM KNOz per
gram fresh weight per day.

 

 

 

 

Temperature Day

Treatment 5th 6th 7th Average
10°C 51.0 54.0 70.0 58.3 a b
15°C 73.5 53.5 43.5 56.8 a b
20°C 73.0 63.0 63.5 66.8 a
25°C 44.5 47.5 44.0 45.3 b
30°C 52.0 42.0 39.0 44.3 b
35°C 13.5 13.0 20.0 15.5

 

Those values containing the same letter are not signif-
icantly different at the 1% level (Duncans multiple range
test).

41

Appendix Table 2.--The nitrate reductase activity of
bermudagrass expressed as uM KN02 per

gram fresh weight per hour.

 

 

 

Temperature Day

Treatment 5th 6th 7th Average
15°C 49.0 36.0 28.5 37.8 b
20°C 81.5 79.5 75.0 78.7
25°C 46.5 44.5 42.0 44.3 b
30°C 51.0 36.5 33.5 40.3 b
35°C 27.5 39.0 49.5 38.7 b
40°C 36.0 37.0 34.0 35.7 b

 

 

Those values containing the same letter are not signif-
icantly different at the 1% level (Duncans multiple range
test).

42

Appendix Table 3.--The nitrate reductase activity per se of
bentgrass and bermudagrass expressed—as
uM KN02 per gram fresh weight per hour.

 

 

 

 

Temperature Bentgrass Samples Bermudagrass Samples
Treatment I II Average I II Average
10°C 9 9.5 9.25 11.5 9.5 10.5
20°C 23.5 21.5 22.5 26.0 23.5 24.75
30°C 35.5 29.0 32.5 38.0 35.5 36.75

40°C 10 9.5 9.75* 36.5 36.0 36.25*

 

*Statistically significant difference [LSD (p = .01) =
20.18] between species is found at this temperature.

43

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