EFFECTS OF NITROGEN NUTRITION AND
TEMPERATURE ON THE GROWTH, CARBOHYDRATE
CONTENT, AND NITROGEN METABOLISM
OF COOL - SEASON GRASSES

Thesis for theDegree of Ph. D.
MICHIGAN .STATE UNIVERSITY
HARLAN R. STOIN
1968

 

Iq-W"

0-169

wk

LIBRARY
Michigan State
S University

  

   

This is to certify that the
thesis entitled

EFFECTS OF NITROGEN NUTRITION AND TEMPERATURE ON
THE GROWTH, CARBOHYDRATE CONTENT, AND NITROGEN
METABOLISM OF COOL-SEASON GRASSES

presented by

Harlan R. Stoin
has been accepted towards fulfillment

of the requirements for

Ph.D. degree in Crog Science

Major professor

{7% We 14- firm/DI)

DateQ’Lvé" A?) A7637
y y

 

 

 

ABSTRACT

EFFECTS OF NITROGEN NUTRITION AND TEMPERATURE ON
THE GROWTH, CARBOHYDRATE CONTENT, AND NITROGEN
METABOLISM OF COOL-SEASON GRASSES

by Harlan R. Stoin

The effects of ammonium vs. nitrate nitrogen and
level of nitrogen on the growth and chemical composition
of several cool—season grasses were studied at day/night
temperatures of 21/16 C and 32/26 C in controlled environ-
ment chambers.

An interaction between nitrogen nutrition and
temperature on the growth of the cool-season grasses
occurred. In a nutrient solution experiment the growth
of Italian ryegrass (Lolium multiflorum Lam.) was always
reduced by the high temperature, and the reduction was
greater at high levels of nitrogen and with ammonium-N.

At the low temperature an increase in level of nitrate—N
increased growth while an increase in level of ammonium-N
decreased growth. In a soil experiment in which nitrifi-
cation of added ammonium-N was not prevented, additions of
either ammonium or nitrate-N caused an increase in growth
of the tops of perennial ryegrass (Lolium perenne L.) and

tall fescue (Festuca arundinacea Schreb.) at the low tem-

 

perature but not at the high temperature. In a second soil

Harlan R. Stoin

experiment in which nitrification of added ammonium-N was
prevented, ammonium-N was superior to nitrate-N for top
growth of Italian ryegrass. An increase in growth for the
high level of ammonium-N but not for the high level of
nitrate-N occurred at the low temperature. No response

to source or level of nitrogen occurred at the high tem—
perature.

Changes in soluble carbohydrate content did not ap-
pear to be the causal factor of growth reduction at high
temperatures nor for the interactions which occurred. In
no cases were the soluble carbohydrates exhausted, nor did
they appear to be present in concentrations limiting to
growth.

In the nutrient solution experiment protein nitrogen
content appeared to be slightly increased by ammonium—N
compared to nitrate—N and slightly decreased by high tem-
perature. The soluble amino nitrogen content was generally
higher with ammonium—N than nitrate—N and increased at the
high temperature with ammonium-N but not with nitrate-N.

In the first soil experiment when no nitrogen was
added, protein nitrogen content at the low temperature was
generally lower than at the high temperature. When nitro—
gen was added, no differences due to temperature occurred
at the first harvest. Between harvests protein nitrogen
content decreased where growth continued but remained the

same in the roots at the high temperature where no growth

Harlan R. Stoin

was occurring. The soluble amino nitrogen content was
generally higher with nitrogen additions and at the high
temperature. The level of available nitrogen appeared to
exert a greater effect than did temperature. Between har-
vests the soluble amino nitrogen content decreased at both
temperatures. In the second soil experiment where free
ammonium, nitrate, glutamine, and asparagine nitrogen were
determined, these fractions all were quite low and in-
creased with an increase in ammonium nitrogen level but
not with an increase in temperature.

The effects of nitrogen nutrition and temperature on
nitrogen metabolism appeared more likely to involve a
blockage or slow down of protein synthesis rather than an
increase in protein breakdown. An accumulation of some
toxic nitrogen compound at high nitrogen levels could also

be involved.

EFFECTS OF NITROGEN NUTRITION AND TEMPERATURE ON
THE GROWTH, CARBOHYDRATE CONTENT, AND NITROGEN

METABOLISM OF COOL-SEASON GRASSES
By

Harlan R; Stoin

A THESIS

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

DOCTOR OF PHILOSOPHY
Department of Crop Science

1968

ACKNOWLEDGMENTS

The author wishes to express his appreciation to
Dr. J. B. Beard for his guidance, criticism, and help
during the course of this study and the preparation of
the manuscript.

Appreciation is also expressed to Drs. F. C.
Elliott, A. E. Erickson, C. M. Harrison, and A. R.

Wolcott for serving on the guidance committee.

ii

TABLE OF CONTENTS

Page
ACKNOWLEDGMENTS . 11
LIST OF TABLES iv
INTRODUCTION 1
REVIEW OF LITERATURE 3
MATERIALS AND METHODS 13
Experiment 1 . . . . 13
Extraction Procedure . . . 15
Soluble Carbohydrate Determination. . . . 16
Soluble Amino Nitrogen Determination . . . . 16
Protein Nitrogen Determination . . . . . . 17
Experiment 2 . . . . . . . . . . . 18
Experiment 3 . . . . . . . . . . 19
Extraction Procedure . . . . . . . 21
Free Ammonium Nitrogen Determination . . . . 23
Glutamine, Asparagine, and Nitrate Nitrogen
Determinations. . . . . . . . . 24
Total Amino Nitrogen Determination. . . . 26
RESULTS. . . . . . . . . . . . . . . 28
Experiment 1 . . . . . . . . . 28
Soluble Carbohydrate Content. . . . . . . 29
Portein Nitrogen Content . . . . . . . . 3O
Soluble Amino Nitrogen Content . . . . . . 31
Experiment 2 . . . . . . 33
Alcohol Soluble Carbohydrate Content . . . 36
Protein Nitrogen Content . . . . . . . 36
Soluble Amino Nitrogen Content . . . . Al
Experiment 3 . . . . . . AA
Water Soluble Carbohydrate Content. A6
Total Amino Nitrogen and Protein Nitrogen Content. A6
Soluble Nitrogen Fractions . . . . . . . . A6
DISCUSSION OF RESULTS . . 53
CONCLUSIONS . 59
BIBLIOGRAPHY . . . . . 62

iii

Table

LIST OF TABLES

The composition of the nutrient solutions
used in Experiment 1

Nutrients added for the various treatments
of Experiment 3 . . . .

The effects of nitrogen nutrition and
temperature on the growth of Italian
ryegrass grown in sand culture. . . .

The effects of nitrogen nutrition and
temperature on the 70% ethanol soluble
carbohydrate content of Italian ryegrass
grown in sand culture. . . . . . . .

The effects of nitrogen nutrition and
temperature on the protein nitrogen content
of Italian ryegrass grown in sand culture

The effects of nitrogen nutrition and
temperature on the soluble amino nitrogen
content of Italian ryegrass grown in
sand culture. . . . . . .

The effects of added nitrogen and temperature
on the growth of perennial ryegrass and
tall fescue grown in soil in a growth
chamber, first harvest . . . . .

The effects of added nitrogen and temperature
on the regrowth and root production of
perennial ryegrass and tall fescue grown
in soil in a growth chamber, second
harvest . . . . . . . . . . . .

The effects of added nitrogen and temperature
on the 70% ethanol soluble carbohydrate
content of perennial ryegrass and tall
fescue grown in soil in a growth chamber,
first harvest

iv

Page

1A

22

28

29

3O

32

3A

35

37

Table Page

10. The effects of added nitrogen and temperature
on the 70% ethanol soluble carbohydrate
content of perennial ryegrass and tall
fescue grown in soil in a growth chamber,
second harvest . . . . . . . . . . 38

11. The effects of added nitrogen and temperature
on the protein nitrogen content of
perennial ryegrass and tall fescue grown
in soil in a growth chamber, first
harvest . . . . . . . . . . . . 39

12. The effects of added nitrogen and temperature
on the protein nitrogen content of
perennial ryegrass and tall fescue
grown in soil in a growth chamber,
second harvest . . . . . . . . . . A0

13. The effects of added nitrogen and temperature
on the soluble amino nitrogen content of
perennial ryegrass and tall fescue grown
in soil in a growth chamber, first
harvest . . . . . . . . . . . . A2

1A. The effects of added nitrogen and temperature
on the soluble amino nitrogen content of
perennial ryegrass and tall fescue grown
in soil in a growth chamber, second
harvest . . . . . . . . . . . . A3

15. The effects of nitrogen nutrition and
temperature on the top growth and water
soluble carbohydrate content of the tops
of Italian ryegrass grown in a soil-sand
mixture in a growth chamber. . . . . . A5

16. The effects of nitrogen nutrition and
temperature on the soluble solids in
the tops of Italian ryegrass grown in
a soil-sand mixture in a growth chamber. . A7

17. The effects of nitrogen nutrition and
temperature on the total amino nitrogen and
protein nitrogen content of the tops of
Italian ryegrass grown in a soil-sand
mixture in a growth chamber. . . . . . A8

18. The effects of nitrogen nutrition and
temperature on the soluble amino nitrogen
fraction in the tops of Italian ryegrass
grown in a soil-sand mixture in a
growth chamber . . . . . . . . . . A9

Table Page

19. The effects of nitrogen nutrition and
temperature on the free ammonium
nitrogen and nitrate nitrogen fractions
in the tops of Italian ryegrass grown in a
soil-sand mixture in a growth chamber . . 51

20. The effects of nitrogen nutrition and
temperature on the glutamine nitrogen
and asparagine nitrogen fractions in
the tops of Italian ryegrass grown in
a soil-sand mixture in a growth
chamber . . . . . . . . . . . . 52

vi

INTRODUCTION

Temperature is a key factor in the growth of grasses
whether they are grown for forage, ornamental, or recrea-
tional use. The production and vigor of the cool—season
grasses are often limited by high temperatures in mid-
summer. Considerable information is available on the
Optimum temperatures for the growth of a number of cool-
season grasses and their response to temperatures above
the optimum. However, little is known of the actual bio—
chemical basis for the reduction of growth of cool-season
grasses at high temperatures.

Nitrogen is also an important factor in the growth
of grasses. Nitrogen has a key role in plant metabolism,
being a major constituent of proteins, enzymes, chloro—
phyll, and nucleic acids. Grasses are quite responsive to
nitrogen fertilization with nitrogen fertility often be-
coming a limiting factor to increased grass growth. Also
an interaction of temperature and nitrogen fertility on
the growth response of grasses occurs. In some cases
reduction in growth at high temperatures was found to be
greater at high nitrogen levels than at lower levels. But
again the actual biochemical basis for this interaction is

unknown.

Many early workers attributed these effects to a
depletion of carbohydrates. High temperatures were hy-
pothesized to cause a reduction in carbohydrates due to
a lower temperature optimum for photosynthesis than for
respiration. High nitrogen levels would reduce carbohy-
drates by utilizing them as carbon skeletons for the
formation of nitrogen-containing compounds. Although
there is evidence that carbohydrates are sometimes reduced
under these conditions, the evidence that carbohydrates
actually become limiting to growth is inconclusive and
unresolved.

Less attention has been given to the effects of en-
vironment on nitrogen metabolism. However, a number of
reports have shown that various nitrogen fractions are
responsive to environmental factors and thus could be
involved in the biochemical reactions that bring about the
reduction in grass growth at high temperatures.

The purpose of this study was to determine the ef—
fects of nitrogen source and level on the growth of cool-
season grasses at the Optimum temperature and at a higher
temperature. Another objective was to determine what ef-
fect these variables would have on the soluble carbohydrate

conterm and on the content of several nitrogen fractions.

REVIEW OF LITERATURE

Levitt (18) distinguishes between direct high temper—
ature injury and indirect injury. Direct high temperature
injury occurs at extremely high temperatures, occurs
quickly, and usually results in death to the plant or to
the affected parts of the plant. Indirect injury occurs
at lower temperatures and does not cause direct kill, al-
though the plants or plant parts may die after a prolonged
period at these temperatures. The separation of these
types of injury is sometimes characterized by a break in
the time versus temperature curve. Levitt further divides
indirect injury into metabolic high temperature injury and
transpirational high temperature injury. This review will
be mainly concerned with metabolic high temperature injury
that occurs at temperatures above the optimum but below the
point of direct high temperature kill.

The optimum temperature for growth has been defined
as the highest temperature at which there is no time factor
operating as distinguished from a maximum-rate temperature
at which growth attains its highest rate (17). Due to
environmental and genetic variables the optimum is really
a range rather than a single temperature and varies for

different plant parts and functions.

The optimum temperatures for growth of a number Of
grass species have been determined. Mitchell (19) found
that the optimum temperature for daily increase in dry
weight Of four cool-season grasses, perennial ryegrass

(Lolium perenne L.), orchardgrass (Dactylis glomerata L.),

 

colonial bentgrass (Agrostis tenuis Sibth.), and velvet—

 

grass (Holcus lanatus L.) was 20 C. In contrast the opti—

 

mum for Dalligrass (Paspalum dilatatum Pois.), a warm-

 

season grass, was near 30 C. Above the Optimum tempera—
tures a rapid decline in growth occurred with growth of
the cool-season grasses ceasing above 35 C. The production
of new tillers was also decreased above 28 C.

An Optimum temperature near 20 C was reported for

perennial ryegrass by Sullivan and Sprague (28) and for top

growth of Kentucky bluegrass (Poa pratensis L.) by Brown (5)

 

and Harrison (12). The optimum temperature for root and
rhizome growth of Kentucky bluegrass was near 16 C.

Several workers have noted that the amount of growth
reduction at temperatures above the optimum is affected by
the level and form of nitrogen applied. Harrison (12)
found that cutting back the leaves of non-vegetative Ken-
tucky bluegrass plants supplied with a minus—nitrogen
nutrient solution, and which had a large quantity of stor-
age rhizomes, was less harmful during the hot summer months
than was short cutting of vegetative plants which received

a continuous supply of nitrogen and which had a smaller

quantity Of storage rhizomes. He also found that after
several defoliations, cultures at 26.A C supplied with
nitrOgen produced no more tOp growth than minus-nitrogen
cultures. In another experiment ammonium nitrogen was
found to produce less growth of tops, roots, and rhizomes
than did nitrate nitrogen when used in nutrient solution
cultures.

Darrow (7) studied the growth of Kentucky bluegrass
in sand culture with ammonium versus nitrate nitrogen, at
pH's of A.5, 5.5, and 6.5, and at soil temperatures of 15,
23, and 35 C. Growth of shoots and roots was reduced with
ammonium nitrogen at all temperatures and pH's. With
either nitrogen source growth was reduced at 35 C compared
to the lower temperatures; however, the reduction was
greater with ammonium nitrogen. The growth reductions
with ammonium nitrogen were greater at low pH's.

Sprague (25) also studied the effects of ammonium
versus nitrate nutrition on colonial bentgrass in sand
culture. Ammonium nitrogen reduced growth of tops and
roots in comparison to nitrate. He conducted no experi-
ments in which temperatures were compared.

Pellett and Roberts (20) found that Kentucky blue-
grass turf grown in solution culture at a low nitrogen
level was more resistant to high temperatures than when

grown at a high nitrogen level.

All of the experiments in which a nitrogen level-
temperature interaction were noted were conducted with
solution cultures. Comparable results from soil or
field experiments have not been reported.

Many workers have attributed the reduction in growth
at high temperatures to carbohydrate depletion brought
about by the lower temperature optimum for photosynthesis
than respiration. High nitrogen levels are also believed
to reduce carbohydrates by rapidly using up the carbohy-
drates as carbon skeletons for nitrogen-containing com—
pounds.

Sullivan and Sprague (28) reported the changes in
carbohydrate levels in perennial ryegrass following clip-
ping at night/day temperatures of 10/15.6, 15.6/21.l,
21.1/26.7, and 26.7/32.2 C. The sucrose content of the
stubble reached a low point two weeks after clipping
followed by a rise in content which was greatest at the
lowest temperature treatment. The decline in sucrose in
the roots lasted four weeks, followed by a rise which oc-
curred only at the lowest temperature treatment. Reducing
sugars fell more gradually after clipping and showed little
response to temperature. The content of fructosan, an
important reserve carbohydrate, was about 25% of the stub-
ble dry weight at the time of clipping. Twenty-eight days
after clipping, the content of fructosan had decreased to

18% at the lowest temperature treatment and 7% at the

highest temperature treatment with intermediate values for
the intermediate temperatures. The content Of fructosan
then began increasing at the two lowest temperature treat—
ments but continued to decrease at the two highest temper—
ature treatments. The fructosan in the roots followed a
similar pattern but at an initial level of 7%.

Differences in the dry weight production Of the re—
growth of tops at the different temperature treatments were
noticeable two weeks after clipping. This was at a time
when the content of fructosan, even at the highest tempera-
ture treatment, was still nearly 8% in the stubble and 2%
in the roots. Although the decline in fructosan continued
at the highest temperature treatment, complete exhaustion
of carbohydrates was not observed at the termination of the
experiment A0 days after clipping.

Brown (5) reported that storage of carbohydrates in
Kentucky bluegrass occurred in spring and autumn when
temperatures were low, but a net loss Of carbohydrates
occurred during late spring and summer when higher tempera-
tures prevailed.

The application of nitrogen fertilizers has also been
shown to reduce carbohydrate levels (29). However, the
effect of high nitrogen levels on carbohydrates is not
clearcut since high nitrogen levels have been found to

increase carbohydrate levels under some conditions (A).

The evidence is not clearcut that reduced carbohy-
drates are the causal factor in reduced growth at high
temperatures. Beinhart (3) studied the growth of white

clover (Trifolium repens L.) in growth chambers and in

 

the field. High temperatures reduced branching or pro-
duction of secondary stolons, a factor closely related
to continued growth and survival of white clover. During
the warm months of June through September, free sugars in
the stolons decreased followed by an increase in October.
Branching was reduced as the decline in sugars was occur—
ring; however, an increase in branching began again in
September before the increase in sugars began. This se—
quence together with some unpublished growth chamber study
data led the author to conclude that carbohydrate supply
was not the limiting factor to summer branching of white
clover.

Duff (8) found that the water soluble and alcohol
soluble carbohydrates were not reduced in leaf tissue of

creeping bentgrass (Agrostis palustris Huds.) grown at

 

supraoptimal temperatures in comparison to those at the
optimum and were increased at a day/night temperature of
AO/30 C.

Green (10) found that carbohydrate levels were re-
duced at high temperatures and high nitrogen levels in four
cool-season grasses, but he concluded that at no time were

carbohydrate levels inadequate for growth.

Tissue culture studies have also shown that factors
other than carbohydrate depletion are involved in high
temperature effects. Tissue cultures of wheat (Triticum

vulgare L.) and tomato (Lycopersicum esculentum L.) roots

 

grown in media containing dextrose, a carbohydrate source,
produced typical temperature versus growth curves with an
Optimum temperature near 30 C (3A, 35).

Several workers have suggested areas other than
carbohydrate depletion that may be involved in high tem-
perature effects. Steward (27) has suggested that an impor-
tant site where environmental factors may interact with
metabolic processes is at the point of contact between
carbohydrate and nitrogen metabolism. Specifically in-
volved would be the keto acids, amino acids, and amides.

In a review of the biochemical aspects of tempera-
ture effects Langridge (15) listed five possible causes
for high temperature effects. All ultimately involved
blockage of synthesis or acceleration of breakdown of
some essential metabolites. Several of the possible causes
involved enzymes and/or amino acids and would thus involve
protein or nitrogen metabolism.

Various nitrogen fractions have been reported to be
affected by environmental factors, including temperature.
Sullivan and Sprague (28), whose results with carbohydrate
changes in perennial ryegrass after clipping were reported
previously, also found changes in several nitrogen frac—

tions. The 80% ethanol soluble nitrogen content in the

lO

tOps as a proportion Of the total nitrogen content
increased following clipping. The largest increase
occurred at the highest temperature treatment. Total
nitrogen in the tops declined with time after clipping

but the decline was markedly less at the highest temper-
ature treatment. Little change in total nitrogen was
noted in the stubble and roots except for an increase at
the highest temperature treatment. Insoluble nitrogen

was also higher in both the stubble and roots at the high-
est temperature treatment. The authors suggested the pos-
sibility of ammonium toxicity due to a rapid digestion of
proteins at high temperatures. Such a possibility had
also been raised by Altergott (1), who worked at tempera-
tures in the range of A0 to 50 C.

More recently, Petinov and Molotkovskii (21, 22)
reported an accumulation of ammonia in several plant spe-
cies grown at high temperatures in the range of A5—60 C.
They were able to partially overcome heat injury by
sprinkling plants with organic acids, which they assumed
neutralized the ammonia and provided energy sources for
metabolizing it. This sprinkling treatment reduced ammonia
levels and increased amide levels. Respiration inhibitors
were reported to decrease heat resistance. They concluded
that the essence of protective reactions of plants to high
temperatures is resynthesis of proteins destroyed by high
temperature. The source Of active metabolites and energy

for resynthesis is respiration.

ll

Beard and Daniel (2) studied the seasonal variation
in the total, nonprotein, glutamine, asparagine, and total
amide nitrogen fractions of Agrostis palustris Huds. leaf
tissue. Temperature was found to be the major environmen-
tal factor influencing seasonal variations in these frac—
tions. Total nitrogen content decreased at temperatures
above 2A C. Glutamine showed the greatest response to
temperature, drOpping to very low values at soil tempera-
tures above 2A C. Asparagine responded much less to
temperature.

Steward (27) has summarized several cases where he
and his co-workers noted effects of environmental factors
on the level and composition of the soluble nitrogen frac-
tion. Studies with the mint plant (Mentha piperita L.)
revealed that daylight, long days, and a high K to Ca
ratio in the nutrient medium tended to promote protein
synthesis and glutamine accumulation in the leaves, espec-
ially at low night temperatures. In contrast, darkness,
short days, a high Ca to K ratio, and high night tempera-
tures tended to favor asparagine accumulation.

The banana (Muga species) variety Gros Michel was
found to have more amide nitrogen with a higher proportion
Of glutamine in fruits that matured in July in Honduras
than in fruits that matured in December, which had pre-
dominantly asparagine. These differences were attributed

mainly to differences in night temperature.

12

In tulip (Tulipa gesneriana) leaves high temperatures
caused an increase in serine/glycine, asparagine, gluta-
mine, gamma—methyleneglutamic acid, and ammonia and a

decrease in aspartic and glutamic acids.

In peas (Pisum sativa L.) grown under long days,

 

the content of asparagine increased with high night tem-

perature.

MATERIALS AND METHODS

Experiment 1. Experiment 1 was designed to determine if

 

ammonium and nitrate nitrogen would have the same effects
on growth at low and high levels in a nutrient solution
and if there would be an interaction with temperature.
Another objective was to determine how the soluble carbo-
hydrate content, protein nitrogen content, and soluble
amino nitrogen content would be affected by these vari-
ables.

Italian ryegrass (Lolium multiflorum Lam. var.

 

MSU—3—LM) was grown in nutrient solution in a growth
chamber at two temperatures and with two sources and two
levels of nitrogen.

One-half gram of seed was planted in white quartz
sand in a 32-ounce waxed cottage cheese container with
drainage holes punched in the bottom. The seed was
lightly covered with sand and placed in a growth chamber
at a day/night temperature Of 21/16 C and a light inten-
sity of 21,500 lux. The light was provided by a combina-
tion of fluorescent and incandescent sources. The day-
length was 16 hours.

The cultures were watered with tap water for 7 days

at which time the young plants were about 1 cm in height.

13

lA

At that time half Of the cups were placed in a growth
chamber at 32/26 C and the other half retained at 21/16 C.
Nutrient solution treatments were initiated the same day.
Two levels of nitrate nitrogen and two levels of ammonium
nitrogen were compared at each temperature. The compo-
sition of the nutrient solution for each nitrogen treat-
ment is shown in Table 1. Each treatment was duplicated.
Approximately 200 ml of fresh nutrient solution was added
twice daily to keep the nutrient solution composition
fairly constant. Drainage from the cups indicated that
this amount of nutrient solution was sufficient to satu-
rate the sand at each watering.

TABLE l.--The composition of the nutrient solutions used
in Experiment 1.

 

Millimoles/liter

 

Nutrient
Solution KH2POA MgSOu Ca(NO3)2 (NHA)2SOA CaSOu pH

 

Low
Nitrate 2.0 1.5 2.5 —— -— 7.98
High
Nitrate 2.0 1.5 10.0 -- -- 7.A7
Low
Ammonium 2.0 1.5 -- 2.5 2.5 7.33
High
Ammonium 2.0 1.5 -- 10.0 10.0 7.80

 

Note: In addition to the above, each solution contained
5 mg/l of Fe as ferric citrate + l ml/l of a solution con-
taining 0.6 g H 803, 0.A g MnCl-AH20, 0.05 g CuSOu-5H20,
and 0.02 g H2Mo A' H20 per liter.

15

The tops and roots were harvested 18 days after the
nitrogen and temperature treatments were initiated (25
days from planting). The roots were freed of sand by
thorough washing with tap water. The plant material was
immediately frozen, freeze—dried, weighed, separated into
tOps and roots, and ground through a 20—mesh screen in a

Wiley mill.

Extraction Procedure. The ground, dried plant material

 

was extracted with 70% ethanol as follows: Twenty ml of
70% ethanol was added to a 0.5 g sample of ground tissue
in a 50 ml centrifuge tube. The tube was stoppered and
shaken 15—18 hours, centrifuged at 35000 g for 10 minutes,
and the extract decanted through a funnel containing
Whatman #1 filter paper into a 100 ml volumetric flask.

An additional 20 ml of 70% ethanol was added to the resi-
due in the centrifuge tube and then shaken an additional
3-A hours. Next the uncentrifuged mixture was poured with
washing into the same filter paper and flask as used
before. The residue in the filter paper was washed with

A additional 10 m1 portions of 70% ethanol. The extract
in the volumetric flask was now made up to volume with

70% ethanol. The soluble amino nitrogen and soluble carbo—
hydrate determinations were made on appropriate dilutions
of this extract. The residue in the filter paper was

saved for the protein nitrogen analysis.

16

Soluble Carbohydrate Determination. Alcohol soluble carbo-

hydrates were determined by the phenol—sulfuric acid

colorimetric method (13) on a 1/10 dilution with 70%

ethanol of the above extract. One ml of the diluted

extract was placed in a matched test tube and 1 ml of 5%

phenol added. Then 5 ml of concentrated sulfuric acid

was added rapidly to ensure mixing of air into the solu-

tion.

The tube was allowed to stand 15 minutes, shaken,

and then allowed to cool to room temperature. The color

density in the tube was read at a wavelength of A90 mu

with a Bausch and Lomb Spectronic 20 spectrophotometer

and compared with a sucrose standard curve.

Soluble Amino Nitrogen Determination. The soluble amino

nitrogen content was determined on a l/10 dilution with

70% ethanol of extract by the ninhydrin colorimetric

method of Rosen (23). The following reagents were used:

1.

2.

Stock NaCN: 0.01 M (A90 mg/l).

Acetate buffer: 3600 g NaOAc - 3H20 + 5 1 H20 +
675 ml glacial acetic acid. Make up to 10 l with
H20. Adjust pH to 5.3 to 5.A with sodium
hydroxide or acetic acid.

Acetate—cyanide: 0.0002 M NaCN in acetate
buffer; 2 ml of Reagent 1 made up to 100 ml
with Reagent 2. This reagent is unstable and
must be used within A hours of mixing (16).

Ninhydrin: 3% in Methyl Cellosolve (peroxide
free).

Diluent: isopropyl alcoholzwater (1:1).

17

One ml of the diluted extract was placed in a matched
test tube and l/2 ml of Reagent 3 and 1/2 ml of Reagent A
were added. The mixture was then heated for 15 minutes in
a boiling water bath. Immediately after removal from the
water bath, 5 ml of diluent (Reagent 5) was rapidly added
and the tube vigorously shaken. The tube was allowed to
cool to room temperature and the color density read at a
wavelength of 570 mu with a Bausch and Lomb Spectronic 20
spectrOphotometer. The color density was compared with a
glutamic acid standard curve. If the optical density of
a tube was too high to give a good reading, further 5 ml
portions of isopropyl-water diluent were added until the

density was below 0.75.

Protein Nitrogen Determination. Protein nitrogen was

 

determined on the residue from the alcohol extraction.
The residue and the filter paper containing it were dried
and placed in a 125 ml Erlenmeyer flask.. Fifty ml of

3 N HCl was added and the mixture autoclaved for 6 hours
at 120 C and 15 lbs pressure to hydrolyze the protein to
amino acids (16). The mixture was then cooled, made up
to 200 ml in a volumetric flask, the solids allowed tO
settle out overnight, and 1 ml of the solution diluted to
10 ml with water. The amino nitrogen content of 1 ml of
the diluted solution was determined by Rosen's method.
The amino nitrogen content was equated with protein nitro—

gen content.

l8

Experiment 2. Experiment 2 was designed to determine

 

whether there would be an interaction of nitrogen nutri-
tion and temperature on the growth and chemical composi-
tion Of grasses grown in soil.

Two cool—season grass species, perennial ryegrass
and tall fescue (Festuca arundinacea Schreb. var.
Kentucky 31) were grown in soil in a growth chamber. The
grasses were grown at two temperatures and with three
nitrogen fertility treatments.

One—half gram of seed was planted in Conover sandy
loam soil in a 32-Ounce waxed cottage cheese container
with drainage holes punched in the bottom. The seed was
lightly covered with soil and watered. The cups were
placed in a growth chamber at 21/16 C day/night tempera-
ture and 21,5000 lux light intensity. The light was pro-
vided by a combination of fluorescent and incandescent
sources. The daylength was 16 hours. After 10 days,
when the young plants were 1-2 cm tall, half of the cups
were placed in a growth chamber at 32/26 C and half
retained at 21/16 C. Four days later three fertility
treatments were applied. The first treatment consisted
of no additional nitrogen, the second treatment was the
addition Of 70 mg of nitrogen per culture as Ca(NO3)2,
and the third treatment was the addition of 70 mg of
nitrogen as (NHA>2SOA° Nothing was added to prevent nitri-

fication Of the added ammonium nitrogen. Each treatment

19

was replicated 3 times. The cultures were kept well
supplied with water.

Eighteen days after the temperature treatments were
initiated (28 days after planting), the top growth above
A cm was harvested from 2 replications Of each treatment.
The tops and roots were harvested from the third replica—
tion. The soil was washed from the roots with tap water.
The plant samples were immediately frozen, freeze—dried,
and ground through a 20-mesh screen with a Wiley mill.

The 2 replications from which only the tops were
harvested were allowed to regrow for 11 days, and then
both the tops and roots from these plants were harvested
and treated as above.

The extraction procedure and chemical determinations
for this experiment were the same as outlined for Experi-
ment 1 except that for the soluble amino nitrogen deter-
mination 1/2 ml of the undiluted extract to which 1/2 m1
Of H20 was added was used instead of 1 ml of the l/lO

diluted extract. This was necessary due to the lower con—

‘tent Of soluble amino nitrogen in Experiment 2 compared

1:0 Experiment 1.

Ehxperiment 3. Experiment 3 was designed to determine if
tide effects of ammonium and nitrate nitrogen on growth and
Cllemical composition Of grasses and their interaction with

temperature would be the same with grasses grown in soil

20

in which the nitrification of ammonium nitrogen was
inhibited.

Italian ryegrass was grown in a soil-sand mixture
in a growth chamber at two temperatures and with two
sources and two levels of nitrogen.

One-half gram of seed was planted as in Experiment
2. The cultures were placed in the greenhouse for 6 days
and then moved into growth chambers at 21/16 C day/night
temperatures and 21,500 lux light intensity. The light
was provided by a combination of fluorescent and incan-
descent sources. The daylength was 16 hours. Three days
later, when the young plants were 2 to 3 cm in height,
the temperature in one chamber was raised to 32/26 C and
the other retained at 21/16 C. Nutrient treatments con-
sisting of ammonium nitrogen versus nitrate nitrogen and
at two levels of each source were applied the same day.
The treatments are shown in Table 2. The N-Serve
(2-chloro-6-(trichloromethyl pyridine)) (Dow Chemical
(Zompany) added to the ammonium treatments served to pre-
Vnent nitrification of the added ammonium sulfate by
Sealectively inhibiting nitrifying bacteria (9). Two weeks
léiter the nitrogen treatments were repeated but with no
afiidition of the other nutrients or N-Serve. Each treat-

Tneflt was replicated 3 times. The cultures were kept well

Su‘pplied with water .

21

Twenty-seven days after the temperature treatments
were initiated (36 days from planting), the top growth
was harvested at ground level and frozen immediately.

Extraction of the samples was done on undried samples.

Extraction procedure. A small portion of the frozen

 

samples was weighed and dried overnight at 90 C to deter-
mine moisture content and dry weight. The dried sample
was cut into small pieces and used for the total amino
nitrogen determination.

The remainder of the frozen sample was allowed to
thaw and then placed in 2 one-quart polyethylene freezer
bags. The sample in the bags was then pressed between
two blocks of wood in a vise. The plant juice ran out the
opening of the bags into a centrifuge tube. The juice so
collected was centrifuged at 35000 g for 10 minutes.

One ml of the cleared juice was pipetted into a
weighing dish, weighed, and dried at 90 C to determine
soluble solids content and to provide a value for adjust—
ment of chemical composition figures for the juice to a
dry weight basis.

This method of pressing the juice out of a previ-
ously frozen plant sample has been shown to give a sample
Of uniform composition comparable to extracts from dried
material (6, 2A).

The cleared juice sample was heated rapidly to 75 C

on a steam bath and maintained between 75—80 C for 10

22

.QOHOOOAMHLOAC pco>mnd Op mucoepmopp EchoEEm map on
pom pom cocoa was o>nom|z mo soapsaom Edd om m mo HE bosons: oco .moazp OOHHOQm
one; mucoepmonp comonpflc one .6020 maco UOfiHOOm Ohms mpamm oonnp pmpflm one ”ouoz

 

 

 

A2 we ozav o.m I- ao.a mm.m mm.m anacoeea swam+

Az we omv m.m In mm.H mm.m mm.m asflcosea 304+

I- Az we oaav o.m ew.a mm.m mm.m mpmapaz emam+

I- Az we mwv m.m em.a mm.m mm.m mpmceaz soa+

sommAzmzv mflmozvao some: Hog sommmo semapmmne spHHapama
massaso\mmfioeaaaaz

 

.m pcoEHLOOxm no mucoEpmopp

mSOHnm> one pom cocoa

mOQOfimpszun.m mqm<e

23

minutes to precipitate soluble proteins (26). Appro-
priate dilutions of this deproteinated extract were used
for determinations of the soluble constituents.

The water soluble carbohydrates and soluble amino
nitrogen were determined by the method outlined in Experi-

ment 1.

Free Ammonium Nitrogen Determination. Free ammonium nitro-
gen was determined colorimetrically with Nessler reagent
(Sargent Chemical Co.) after the free ammonium ions were
separated from the extract on a 5 X 0.9 cm column of
AG 50-X—A cation exchange resin, 200-A00 mesh in the
sodium form (Calbiochem). Separation of the ammonium ions
from the extract was necessary because of substances in
the pure extract which interfered with direct Nessleriza-
tion.

The exchange column was poured with AG 50-X-A in the
H+ form and then converted to the Na+ form by passing 10
to 15 ml of 2 N NaOH through the column with A to 6 lbs.
of pressure, followed by washing with distilled water. A
1 m1 sample of the undiluted, deproteinated extract was
passed into the column and washed with several portions
Of distilled water. The ammonium ions were retained on
the column. Elution of the ammonium ions was performed by
passing 10 ml Of 1.5 N NaOAC through the column, also with
H to 6 lbs. of pressure. The eluate was collected in a

50 m1 volumetric flask.

2A

Following elution, the solution in the 50 ml volu-
metric flask, which contained the ammonium ions, was made
almost to volume with distilled water. Then 1 m1 of
Nessler reagent was added and the mixture was shaken and
allowed tO stand 15 minutes. After 15 minutes the solu—
tion was made up to volume and a portion poured into a
matched test tube. The color density was read at a wave-
length of A10 mu with a Bausch and Lomb Spectronic 20
spectrophotometer. The optical density was compared with
an NHuCl standard curve. In order to prepare a standard
comparable to the eluate solutions, it was necessary to
add 10 ml of 1.5 N NaOAC to the flasks in which the NHuCl
standards were run since the NaOAC in the eluate solutions
produced a slight cloudiness and thus increased the opti-

cal density in comparison to ammonium ions in water.

Glutamine, Asparagine, and Nitrate Nitrogen Determinations.
Glutamine, asparagine, and nitrate nitrogen were deter—
mined by a combination and modification of the methods of
'Vickery gt_al. (33) and Varner gt_a£. (32).

In order to hydrolyze the amide group of glutamine
to ammonia without hydrolyzing the asparagine, 1 ml of
depncoteinated extract was placed in a 50 ml Erlenmeyer
flask and 3 ml of a phosphate—borate buffer at pH 6.5
(ll).2 g KH2POu and A.8 g NaZBAO7 per 1.) was added and

the Inixture heated for two hours on a steam bath (33).

25

To prevent evaporation and loss of ammonia, the flask was
stoppered with a one-hole rubber stopper into which a
1 ml transfer pipet was fitted.

Following heating, a few drops Of water were washed
down the pipet to return any volatilized ammonia to the
flask. The contents of the flask were cooled and then
transferred with washing to the distillation flask of a
micro—Kjeldahl distillation unit. Ten ml of a saturated
Na2BuO7 solution adjusted to a pH of 10 with A0% NaOH was
added to make the contents slightly alkaline. The ammonia
was then steam distilled into a receiving flask containing
10 ml Of 1% boric acid plus a bromcresol green and methyl
red indicator (20 g boric acid + 10 m1 Of a 0.033%
bromcresol green and 0.066% methyl red in methanol solu—
tion made up to 2 l with water). Distillation was con-
tinued 5 minutes. The ammonia thus collected consisted
of that from free ammonium and glutamine nitrogen.

Next, 10 ml of A0% NaOH was added to the distilla-
tion flask, a new receiving flask containing 10 ml of
boric acid and indicator was put in place, and steam dis-
tillation continued for 3 minutes. The ammonia now col—
lected was from the hydrolysis of the amide group of
asparagine under these strongly alkaline conditions (32).

Nitrate nitrogen was also determined on the same

sample after the nitrate was reduced to ammonia (32).

First glucose interference was removed by adding 2 ml of

26

15% tartaric acid and 1/2 ml of 25% cupric sulfate to the
distillation flask. Then the mixture was steam heated
for 3 minutes. Next, a new receiving flask containing 1%
boric acid and indicator was put in place and 5 ml of 20%
ferrous sulfate was added to the distillation flask. The
ferrous sulfate reduced the nitrate to ammonia, which was
steam distilled into the receiving flask for 3 minutes.
This procedure also reduces nitrite to ammonia, but
nitrite is usually present only in very small quantities
in plant tissue.

The boric acid in the receiving flasks was titrated
with 0.0076 N HCl to a pink end point. A blank was run
through all three distillations and the value of the

blank subtracted from the sample values.

Total Amino Nitrogen Determination. Total amino nitrogen
was determined on an over—dried sample which had been cut
into fine pieces. About 100 mg of dried sample was placed
in a 100 ml volumetric flask and 10 ml of A N HCl added.
The flask was then autoclaved at 120 C and 15 lbs. pressure
to hydrolyze the protein to amino acids. After 1 hour the
flask was removed from the autoclave, the contents were
SWirled around to wash down the sides, and then autoclaved
for*three more hours. Next, 10 ml of additional A N HCl
Was added to wash the sides down, and autoclaving was con-
tinued for two additional hours. Following autoclaving,

the flask was filled to volume with water and the solids

27

allowed to settle out overnight. The amino nitrogen con-
tent Of a one ml sample of a 1/10 dilution with water of
this solution was then determined by Rosen's method as
outlined for Experiment 1. Amide nitrogen, which is
hydrolyzed to ammonium nitrogen by this process, is also

included in the amino nitrogen determination.

RESULTS

The results of the experiments were analyzed statis-
tically as randomized block experiments. The treatment
means were separated by using Duncan's multiple range test.
Means in the tables followed by an identical letter do
not differ at the 5% level of significance. The tops and

roots were analyzed separately.

Experiment 1. The growth of Italian ryegrass in nutrient

 

solution was affected by nitrogen source, nitrogen level,
and temperature (Table 3). Significant interactions among

the three variables also occurred.

TABLE 3. The effects of nitrogen nutrition and temperature
on the growth of Italian ryegrass grown in sand culture
(means of 2 replications). .

Day/Night Temperature

 

Nutrient Solution

 

 

Variable 21/16 C 32/26 C
Tops Roots Tops Roots

g/pot g/pot
Low Nitrate 2.5de 1.5d 1.10 1.00
Higfld Nitrate 3.2f 2.6e 0.7b 0.6b
Imnv.Ammonium 2.6e 1.5d 0.7b 0.5b
High Ammonium 2.Ad 1.00 0.1a 0.1a

 

 

29

At the low temperature and low nitrogen level
nitrate—N was equal to ammonium-N as a nitrogen source.
However, nitrate-N increased growth whereas ammonium—N
decreased growth at the high level of nitrogen. Tops and
roots reacted similarly.

Growth of tops and roots was reduced for all nitro—
gen treatments at the high temperature in comparison to
the same treatment at the low temperature. This reduction
was greater at high nitrogen levels and was greater with

ammonium-N than with nitrate—N.

Soluble Carbohydrate Content. The effects Of nitrogen

 

treatment and temperature on the 70% ethanol soluble

carbohydrate content are shown in Table A.

TABLE A. The effects of nitrogen nutrition and temperature
on the 70% ethanol soluble carbohydrate content of Italian
ryegrass grown in sand culture (means of 2 replications).

 

Day/Night Temperature

 

Nutrient Solution

 

 

Variable 21/16 C 32/26 C
Tops Roots Tops Roots
mg/g DW mg/g DW
Low Nitrate 89bc A6cd 92cd 3Aab
High Nitrate lOOd A6cd 76a AOde
Low Ammonium 90bc A8d 87bc 37bc

High Ammonium 81ab A3bcd 9Acd 26a

g

30

At the low temperature the only nutritional response
found was a small increase in carbohydrate in the tops for
the high nitrate-N treatment. High temperature caused no
change in the carbohydrate content in the tops for the low
level of either nitrate—N or ammonium-N. However, the
content for the high nitrate—N treatment was lower at the
high temperature than at the low temperature and lower
than for the other nitrogen treatments at the high tempera-
ture. The content for the high ammonium-N treatment was

higher at the high temperature than at the low temperature.

Protein Nitrogen Content. The effects of nitrogen treat-
ment and temperature on protein content are shown in

Table 5.

TABLE 5. The effects of nitrogen nutrition and temperature
on the protein nitrogen content of Italian ryegrass grown
in sand culture (means of 2 replications).

 

Day/Night Temperature

 

Nutrient Solution

 

 

Variable 21/16 C 32/26 C
Tops Roots Tops Roots
ma N/g DW mg N/g DW
Low Nitrate 23.30abc 12.72cd 20.16ab 8.2Aab
High Nitrate 25.5Aabc 10.02abc 19.26a 10.56bc
Low Ammonium 27.33bc lA.lAd 23.30abc 12.72cd

High Ammonium 30.020 17.25e 19.75a 7.A7a

 

 

31

No differences in the tops due to nitrogen treatment
were found at either temperature. High temperature did
cause a reduction in protein nitrogen content with the
high ammonium—N treatment, but no reduction occurred for
the other treatments, although the content for the low and
high nitrate—N treatments was lower than for the high
ammonium-N treatment, and the high nitrate-N treatment was
also lower than for the low ammonium-N treatment.

In the roots the protein nitrogen content was always
lower than in the tops. It was higher for the high
ammonium-N treatment at the low temperature. The protein
nitrogen content for the low ammonium-N treatment was
significantly higher than for the high nitrate—N treatment
but not significantly higher than for the low nitrate—N
treatment. At the high temperature the protein nitrogen
content of the roots was lower compared to the content at
'the low temperature for the low nitrate-N and high
armonium-N treatments but not for the high nitrate—N and
ilow ammonium—N treatments. At the high temperature the
{Drotein nitrogen content was also higher for the low
aimmonium-N treatment than for the low nitrate-N treatment
twat was lower for the high ammonium-N treatment than for

tune high nitrate-N treatment.

SOluble Amino Nitrogen Content. The effects of nitrogen
treatment and temperature on the soluble amino nitrogen

content are shown in Table 6.

32

TABLE 6.—-The effects of nitrogen nutrition and temperature
on the soluble amino nitrogen content of Italian ryegrass
grown in sand culture (means of 2 replications).

 

Day/Night Temperature

 

Nutrient Solution

 

 

Variable 21/16 C 32/26 C
Tops Roots Tops Roots

ma N/a DW mg N/g 0w
Low Nitrate 2.60a 1.2Aa 2.06a 1.12a
High Nitrate 2.59a 0.9Aa 2.77a l.A8a
Low Ammonium A.65b 5.36bc 6.280 5.900
High Ammonium 7.73d 5.l8b 20.16e 9.78d

 

NO differences due to nitrogen level or temperature
occurred for the nitrate-N treatments. The content of
soluble amino nitrogen in both the tops and roots was
much higher with ammonium—N than with nitrate—N. Also
the high level of ammonium-N and the high temperature
increased the soluble amino nitrogen content of the tops.
lune only increase in the roots due to ammonium level and
tennperature occurred for the high ammonium-N treatment at
thi? high temperature. The increases with ammonium—N
Cornpared to nitrate-N were greater in the roots than in

thee tops except for the ammonium—N treatment at the high

teuiperature.

33

Experiment 2. The growth of perennial ryegrass and tall

 

fescue in soil was also affected by additions of nitrogen
and by temperature. A significant interaction between
nitrogen fertility and temperature also occurred. The

dry weight production above A cm 28 days after planting is
shown in Table 7. The regrowth above A cm after 10 days
is shown in Table 8. The dry weights of the roots for the
one replication harvested at the first date are shown in
Table 7 and for the two replications harvested at the
second date in Table 8.

At the low temperature there was an increase in
growth of the tops with applied nitrogen. Either source
of nitrogen gave the same response. The same was true
for the regrowth at low temperatures. There were no dif-
ferences between species.

At the high temperature there was no response to
added nitrogen of either source at the first harvest nor
for regrowth. With no added nitrogen, the growth Of tops
at the high temperature in comparison to the low tempera-
ture was the same for the tall fescue and only slightly
reduced for the perennial ryegrass. However, growth of
tOps was always significantly reduced by the high tempera-
ture for treatments which received added nitrogen. There
IWEre no differences due to species or nitrogen source.

The roots showed no response to added nitrogen at

Gitdaer temperature but were always greatly reduced by the

3A

TABLE 7.——The effects of added nitrogen and temperature on
the growth of perennial ryegrass and tall fescue grown in
soil in a growth chamber. First harvest.

 

Day/Night Temperature

 

 

 

Fertility Treatment 2l/l6 C 32/26 C
Topsl Roots2 Topsl Roots2
g/pot g/pot

Lolium perenne

 

NO added N 1.00 1.5 0.8ab 0.5
+Ammonium N 1.3d 1.3 0.8abc 0.7
+Nitrate N 1.3d 1.2 0.9bc 0.8

Festuca arundinacea

 

NO added N 0.8abc 1.8 0.7a 0.8
+Ammonium N l.Ad 1.3 0.8abc 0.7
+Nitrate N 1.5d 1.5 0.8abc 0.6

 

lTOngOWth above A cm. Means of 3 replications.

2Values of one replication.

35

TABLE 8.—-The effects of added nitrogen and temperature on
the regrowth and root production of perennial ryegrass and
tall fescue grown in soil in a growth chamber. Second
harvest (means of 2 replications).

 

Day/Night Temperature

 

 

 

 

 

 

Fertility Treatment 21/16 C 32/26 C
Regrowth Regrowth
above Acm ROOtS above Acm ROOtS
g/pot g/pot
Loliumgperenne
NO added N O.Abc 2.0b 0.2a 0.8a
+Ammonium N 0.5cd 2.3bc 0.2a 0.8a
+Nitrate N 0.6d 2.Ac 0.2a 0.7a
iFestuca arundinacea
No added N 0.3ab 2.2bc 0.2a 0.8a
'+Ammonium N 0.50d 2.0b 0.2a 0.8a

+Nitrate N 0.6d 2.0b 0.3ab 0.8a

‘

36

high temperature. There were no species differences.
Although only one sample of roots was available at the
first harvest, the roots appeared to continue growing
between harvests at the low temperature but grew very
little, if at all, between harvests at the high tempera—

ture.

Alcohol Soluble CarbOhydrate Content. The effects of
added nitrogen and temperature on the 70% ethanol soluble
carbohydrate content are shown for the first harvest in
Table 9 and for the second harvest in Table 10.

The 70% ethanol soluble carbohydrate content did not
vary much between treatments or harvest dates. It was
always much lower in the roots than in the tops. A few
differences occurred but did not seem related to any par—
ticular temperature or fertility treatment except that the
carbohydrate content may have been slightly lower at the
high temperature with added nitrogen for the first harvest
Of tall fescue. This difference did not occur at the

second harvest.

Egotein Nitrogen Content. The effects Of added nitrogen
and temperature on the protein nitrogen content are shown
for the first harvest in Table 11 and the second harvest
in Table 12.

At the first harvest and the low temperature the
protein nitrogen content of the tops and roots was higher

witliadded nitrogen. At the high temperature there may

37

TABLE 9.--The effects of added nitrogen and temperature on
the 70% ethanol soluble carbohydrate content of perennial
ryegrass and tall fescue grown in soil in a growth chamber.

First harvest.

 

Day/Night Temperature

 

 

 

 

 

Fertility Treatment 21/16 C 32/26 C
Topsl Roots Topsl Roots2

mg/g DW mg/g Dw

Loliumgperenne

NO added N lOlab A2 106bc 55

+Ammonium N 86ab AA 10Abc AA

+Nitrate N 10200 51 81a 51

Festuca arundinacea

No added N 1110 32 103bc 76

+Ammonium N 108c AA 77a A3

+Nitrate N 110c 36 79a 57

 

1Means of 3 replications.

2Values of one replication.

38

TABLE lO.—-The effects of added nitrogen and temperature on
the 70% ethanol soluble carbohydrate content of perennial
ryegrass and tall fescue grown in soil in a growth chamber.
Second harvest (means of 2 replications).

 

Day/Night Temperature

 

 

 

 

 

 

Fertility Treatment 21/16 C 32/26 C

Tops Roots Tops Roots

mg/g DW mg/g DW

Lolium perenne
No added N 83a 3Aa 97abc A6bcde
+Ammonium N 95abc 39abc 9Aabc 39abc
+Nitrate N lO3abC A0abcd 97abc A3abcde
Festuca arundinacea
No added N 99abc A8cde 98abc 50e
+Ammonium N 108bc 50e lOAabc 50e
+Nitrate N 1150 36ab 9lab A2abcde

 

39

TABLE ll.--The effects of added nitrogen and temperature
on the protein nitrogen content of perennial ryegrass and
tall fescue grown in soil in a growth chamber. First
harvest.

 

Day/Night Temperature

 

 

 

Fertility Treatment 21/16 C 32/26 C
Topsl Roots2 Topsl Roots2
mg N/g DW mg N/g DW

Lolium perenne

 

NO added N 12.6Aa 7.5A 1A.76ab 11.19
+Ammonium N 18.07bc 10.60 18.33bc 11.5A
+Nitrate N 20.160 11.31 19.260 12.25

Festuca arundinacea

 

 

NO added N l3.A9a 6.60 18.88bc 11.31

+Ammonium N 17.97bc 10.13 18.29bc 11.31

+Nitrate N 18.00bC 8.95 20.31C 10.8A
1

Means of 3 replications.

2Values Of one replication.

A0

TABLE 12.-—The effects of added nitrogen and temperature on
the protein nitrogen content of perennial ryegrass and tall
fescue grown in soil in a growth chamber. Second harvest
(means of 2 replications).

 

Day/Night Temperature

 

 

 

 

Fertility Treatment 21/16 C 32/26 C
Tops Roots Tops Roots
ms N/s DW mg N/g 0w

Lolium perenne

 

NO added N 9.5Aab 6.72ab 11.A2abc 9.300d
+Ammonium N 11.78abc 6.83ab 13.3lab 10.60d
+Nitrate N 11.78abc 7.66bc 12.02abc 10.13d

Festuca arundinacea

 

NO added N 8.72a 5.18a l2.A8bc 10.02d
+Ammonium N 11.78abc 6.95ab 12.72bc 11.19d

+Nitrate N ll.l9abc 7.07b l2.A8bc 10.A8d

 

Al

have been a small increase in the tops with added nitrogen
for perennial ryegrass but not for tall fescue. No change
in protein nitrogen content occurred in the roots due to
added nitrogen at the high temperature. However, the
protein nitrogen content of the roots was higher at the
high temperature than at the low temperature when no
nitrogen was added but was not different for treatments
receiving added nitrogen. The protein nitrogen content
between species did not differ except for the one case
noted above.

By the time of the second harvest, the protein
nitrogen levels had decreased from the first harvest for
all treatments except for the roots at the high tempera-
ture. The differences due to added nitrogen at the low
temperature were now no longer statistically significant.
The protein nitrogen contents Of the tops decreased to
the same levels at both temperatures, but in the roots
the protein nitrogen levels decreased to such an extent
at the low temperature than they were now significantly
lower than at the high temperature. There were no dif-

ferences between species at the second harvest.

Soluble Amino Nitrogen Content. The effects of added
nitrogen and temperature on the soluble amino nitrogen
content are shown for the first harvest in Table 13 and

for the second harvest in Table 1A.

A2

TABLE l3.--The effects Of added nitrogen and temperature
on the soluble amino nitrogen content Of perennial rye-
grass and tall fescue grown in soil in a growth chamber.
First harvest.

 

Day/Night Temperature

 

 

 

Fertility Treatment 2l/l6 C 32/26 C
Topsl Roots2 TOpsl Roots2
mg N/g DW mg N/g DW

Lolium perenne

 

NO added N 1.01ab 0.9A 1.A3Cd 1.20
+Ammonium N 1.29bc 1.18 1.78def 1.3A
+Nitrate N 1.83ef 1.18 1.82ef 1.32

Festuca arundinacea

 

 

NO added N 0.90a 0.90 1.A7Cde l.Al

+Ammonium N 1.38bc 0.90 1.82ef 1.77

+Nitrate N 1.37bc 0.78 1.99f 1.88
1

Means of 3 replications.

2Values of one replication.

A3

TABLE 1A.——The effects of added nitrogen and temperature
on the soluble amino nitrogen content of perennial rye-
grass and tall fescue grown in soil in a growth chamber.

Second harvest (means of 2 replications).

 

Day/Night Temperature

 

 

 

 

 

Fertility Treatment 21/16 C 32/26 C

Tops Roots Tops Roots
ma N/a DW mg N/g DW

Loliumgperenne

No added N 0.56ab 0.28b 0.78cd 0.67c

+Ammonium N 0.67bc 0.33b l.l2e 0.88d

+Nitrate N 0.65bc 0.27b 1.26f 0.88d

Festuca arundinacea

No added N 0.A6a 0.15a 0.90d 0.78d

+Ammonium N 0.60ab 0.26b 1.26f 1.06e

+Nitrate N 0.62b 0.22ab l.AAg 0.86d

 

AA

For the first harvest, added nitrogen caused an
increase in soluble amino nitrogen content of the tops
and roots at both temperatures except for the added
ammonium-N treatment of perennial ryegrass at the low
temperature and the roots of tall fescue at the low
temperature. The content was always higher at the high
temperature in comparison to the same treatment at the
low temperature except for the added nitrate—N of peren—
nial ryegrass. The soluble amino nitrogen content of the
roots was generally the same or less than that of the
tops.

At the second harvest added nitrogen caused an
increase in soluble amino nitrogen content of the tops
and roots except for the roots of perennial ryegrass at
the low temperature and the roots of tall fescue for the
added nitrate-N treatment at the high temperature. The
soluble amino nitrogen content had considerably decreased
between harvests for all treatments, more at the low
temperature than at the high temperature. It was always

lower in the roots than the tops.

Experiment 3. The growth of annual reygrass in a soil—

 

sand mixture was also affected by nitrogen source and
level and by temperature (Table 15). Significant inter-
actions among the variables also occurred.

The top growth with ammonium-N was superior to

that with nitrate—N at the low temperature. A response

A5

to the high level of nitrogen occurred with the addition
of ammonium-N but not with nitrate-N. At the high temper-

ature no differences due to added nitrogen occurred.

TABLE 15.—-The effects of nitrogen nutrition and tempera—
ture on the top growth and water soluble carbohydrate con—
tent of the tops of Italian ryegrass grown in a soil-sand
mixture in a growth chamber (means of 3 replications).

 

Dry Weight Soluble
Carbohydrate

 

 

Fertility Treatment
21/16 C 32/26 C 21/16 C 32/26 C

 

g/pot g/pot
+Low Nitrate N 2.9a 3.5abc 19A0 176b0
+High Nitrate N 3.9ab0 3.3ab 2200 lAOab
+Low Ammonium N A.70 3.6abc 2130 1A2ab
+High Ammonium N 6.7d A.Abc l78bc 121a

 

There was little difference between temperature
treatments with added nitrate-N, but growth was signifi-
cantly reduced by the high temperature with added
ammonium—N. Even though no differences in dry weight
production due to temperature occurred for some treat-
ments, the grass was always a darker, bluish—green color
with more dead leaves and leaf tips evident at the high

temperature.

A6

Water Soluble Carbohydrate Content. The effects of the

 

nitrogen treatments and temperature on the water soluble
carbohydrate content are shown in Table 15.

No differences in soluble carbohydrate content
due to nitrogen treatments occurred at either temperature.
However, the soluble carbohydrate content was lower at
the high temperature for all nitrogen treatments except
the low nitrate-N treatment.

The total soluble solids (Table 16) also showed a
decrease at the high temperature. A comparison of the
total soluble solids with the soluble solids minus water
soluble carbohydrates indicated that the temperature
responses of the total soluble solids were due to the

water soluble carbohydrate fraction.

Total Amino Nitrogen and Protein Nitrogen Content. The
total amino nitrogen and protein nitrogen contents were
highly variable and no definite trend due to treatments

was found (Table 17).

Soluble Nitrogen Fractions. Several of the soluble nitro-

 

gen fractions were responsive to nitrogen treatments
and/or temperature.

The soluble amino nitrogen was quite variable in
this experiment (Table 18). At both temperatures the high
level of ammonium—N or nitrate—N increased the soluble

amino nitrogen content but the content did not differ at

A7

 

 

maefi mmfim «mam nfimm z asacoesa smam+
mmwfi Amen mmmm Beam 2 asacoesa soa+
mama mesa msmm nmmm z newspaz nmam+
mmma meow mamm swam z mpmnpflz 304+
30 w\mE 30 w\we
o emxmm o maxam o mm\mm o ma\am

 

OLSOOLOQEOB uanZ\me psoEpmope mpflaflpnom

 

 

monmncmnoonmo OHOSHom nonmz
mscfiz moflaom macsaom

meaaom mansaom Hence

 

.AmCOHOHOHHOon m co mcmmev
penance npsonm m 2H magpxfle ecmmlaflom m cfl csonw mmmpmomn cmfiampH mo mcou one cfl
mcflaom mannaom on» no OOSOHLOQEOO ccm coapfinpsc comospflc CO mpooemo onenl.mH mqm<e

A8

 

.comonpfic oumhpfic pcooxo mCOHpOmne cowogpfic OHQSHOm mSOHnm>
map mscfie cowoapflc ocflem HHpOp one mm UOOOHSOHHO mm; cowonpfic Camponm

H

 

 

nmms.m nmao.fia onmmm.HH onm©.mH z Seacoasa cmam+
nsm.ma nmmm.m om:.ma onnmm.m z egacosea soa+
nmem.oa nnmm.a unmao.mfi nmmm.m z mpmapaz nmam+
nmoa.m mom.e onmms.m mmm.s z newspaz 304+
gm mxz me an m\z me
o mm\mm o ©H\Hm o mm\mm o ©H\Hm

 

endmeOQEoB pQwHZ\me

 

Hz Campopm

semanmmne snaaapnmm

 

z cease fiance

 

.AmQOHmefiHOon m mo mcmoev nonsmno nusonm M GA OASOxHE ccmmuaflom m
CH czonw mmmnwozm :mflampH no onu map mo pcopcoo comoppfic GHOOOOQ one cowonpflc
OCHEO HOOOO map so OLSOOLOOEOO one coapflnpsc comonpflc mo mpooewo OQBII.NH mqm<e

 

‘
_- .

Ln Insulin... I341..—

 

A9

 

 

 

 

mHH.H o:w.o 02:.H UHN.H z EsflcoEE< Lwflm+
Comm.o nmmm.o opmw.o Omm:.o z Esflcoee< 3oq+
em©.o comm.o omw.o opmom.o z mpmnpaz cmam+
ons:.o mom.o Ommm.o ma:.o z Opmnpfiz 30q+
3Q m\z we 3a m\z me

o wm\mm o ©a\am o mm\mm o ©H\Hm

OASOHLOQEoB OhmHZ\zmQ pcoepmone hpflfiflpnom
z ocflmmpmdm< pcm OCHEmpsao z ocfiE< mandaom

mzcflz z OCHE< OHQSHOW

 

“.mcoHOOOHHOOL m mo mcmoEV penance £OSOLm m
CH OLSOxHE ccmmuaflom m CH czonm mmmpwozp cmfiampH mo mOOO one CH coapompu nowoapflc
ocHEm OHOSHom 050 no OLSOHLOQEOO new COHOHOOSQ cowonpflc no mpoomeo oneln.mfi mqm<s

50

the high temperature in comparison to the same nitrogen
treatment at the low temperature.

If the amino nitrogen fraction contributed by the
amides glutamine and asparagine is subtracted from the
total soluble amino nitrogen, an increase at the high
temperature is evident with the exception of the high
nitrate—N treatment (Table 18).

The levels of free ammonium and nitrate nitrogen
in the plant tissue were quite low and quite variable
for all treatments (Table 19). Both of these fractions
generally increased for the high level of nitrogen at
both temperatures although there were a few exceptions.
Temperature had no significant effect on these fractions
except for a slight increase in free ammonium nitrogen
at the high temperature for the low ammonium—N treatment.

Glutamine and asparagine nitrogen levels were also
quite low and quite variable (Table 20). A large increase
in both of these amides did occur for the high ammonium
treatment at both temperatures. NO significant differences

due to temperature treatment occurred.

 

51

. v..-
I‘llililr

 

nmmm pom one: one: 2 azacoesa gmam+

 

 

 

 

mmm mHH berm Odom z Ezficoee< soq+
«am As gem new 2 mpnnpaz cmam+
mma HHH mmm mom 2 mpmnpfiz KOQ+
3o m\z m: 3a m\z ms
0 mm\mm o mH\Hm o mm\mm o ©H\Hm
OLSOOLOOEoB pnwfl2\mmo pcoEmeLB mpflflfippom
z oumnpflz z EsacoEE< mosh

 

.AmCOflmeflHOon m mo memoev nonemno Bazonw Q Ca manpxfie UnmeHHOm m
cfi csopw mmmnwomu CHHHmOH no mcou map OH mCOHuompe cmmoapflc onwnpfic new cowoppflc
EzficoEEm moan one no OLSOmLOOEOO use GOHOHLOSG cowonpw: mo mpoommo 0351:.ma mqm<e

 

 

52

nHmm Beam nomm poem 2 schoee< gmHm+

 

 

 

 

mHmH mmmH HQOH cum 2 EchoEE< SOH+
mmmH mHoH mmmH no: 2 meannHz anm+
mom mHmH woe m mo 2 OpmeHz 3oq+
3a m\z ms 3a m\z m:
o mm\mm o mH\Hm o om\mm o mH\Hm
OHSHOHOOEoB pan2\%mQ psoEHmone HpHHHpHom
z ocmeHMQm< z OQHEOHSHU

 

.AmCOHOOOHHQOH m mo mcmmev nonsmzo gasonm m CH OHSHxHE ecmmnHHom m CH
csonw mmmpwozn cmHHmpH mo meow onp CH mQOHpomnm cowoanc ochmHmcmm use somOHHHc
ocHEwpsHm one no OHSOHHOQEOO new COHOHHOSQ comoanc mo mpoomeo onenn.om mqm<e

 

DISCUSSION OF RESULTS

A definite interaction between nitrogen source and
level and temperature on the growth of the cool-season
grasses in both the nutrient solution and soil experiments
occurred. However, the interaction was not the same for

both types of experiments. In the nutrient solution

 

experiment, growth was always reduced by the high temper—
ature with a greater reduction at the high level of nitro-
gen. On the other hand, growth was reduced only slightly
or not at all at the high temperature and low nitrogen
level in the soil experiments. At the high nitrogen level
growth was reduced at the high temperature compared to the
low temperature although the growth at the high temperature
was not less than that at the low nitrogen level.

One would not necessarily expect the same results
from nutrient solution and soil experiments. Soil has a
buffering capacity for many nutrient ions including
ammonium ions due to ion exchange on charged sites. Thus
the concentration Of ions in the soil solution may be less
than in a nutrient solution yet the ions available to the
plant for growth from the soil system may be as high or
higher. Also there are microbial relations in the soil
that do not exist in the nutrient solution. Another very

important factor is the loss of nitrogen from the soil

53

5A

experiments as nitrogen was utilized by the growing plants
and leached out in drainage water. The nitrogen level in
the soil was probably much lower by harvest time than at
the start of the experiments. The low levels of the
various nitrogen fractions determined in the soil experi-
ments indicated that levels of available nitrogen had

become quite low in the soil experiments. HoweVer, the

‘ ‘ T33“...’n'
’1
I

yields and responses of the grasses to nitrogen in the

 

soil experiments indicated that they were not starved for r-‘
nitrogen during the entire experimental period. In con-

trast to the nitrogen losses with the soil experiments,

the nitrogen levels in the nutrient solution experiment

remained constant throughout the experimental period since

the solutions were replaced daily.

Ammonium-N and nitrate—N produced different effects
on growth. The results from the nutrient solution experi-
ment agreed in part with those of Darrow, Harrison, and
Sprague (7, 12, 25), who found nitrate-N to be superior
to ammonium-N as a nitrogen source for grasses grown in
nutrient solution. EXperiment 1 gave results similar to
theirs at the high level of nitrogen; however, at the low
nitrogen level and low temperature nitrate—N and ammonium-N
were equal as nitrogen sources. In Experiment 3, a soil
experiment, in which nitrification Of ammonium nitrogen
was inhibited, ammonium-N was found to be superior to

nitrate-N for top growth. However, since these cultures

55

were heavily watered, it is possible that this result was
due to greater leaching losses of nitrate—N than ammonium—N
rather than to a specific effect of the ammonium ion.

The content of soluble carbohydrates was somewhat
lower at the high temperature for some treatments, but

this result was not consistent for all treatments and all

 

experiments. In no case was the soluble carbohydrate
exhausted, nor did it appear to be present in concentra-

tions low enough to limit growth. ..

 

Nitrogen metabolism was significantly affected by
both nitrogen nutrition and temperature. In the nutrient
solution experiment there were no significant differences
in protein nitrogen content of the tops due to nitrogen
nutrition. However, there may have been a tendency for
a small increase with ammonium—N compared to nitrate—N
and possibly a small increase with the high level of
nitrogen. There was also some indication of a decrease
in protein nitrogen at the high temperature. Also the
soluble amino nitrogen content generally increased at the
high temperature and at the high nitrogen level with
ammonium-N, but these changes did not occur with nitrate—N.
This result may indicate that some sort of regulation of
the maximum amount Of nitrate-N that can be reduced and
assimilated into organic forms occurs, whereas with

ammonium—N, assimilation continues as long as ammonium

56

ions are taken up. Such a regulation has been observed

in blue—green algae (Chlorella species) (30,31).

 

Increases in soluble amino nitrogen such as occurred
at the high temperature with ammonium-N were Observed by
Sullivan and Sprague (28) and Petinov and Molotkovskii
(21,22). They attributed these increases to increased
protein breakdown without resynthesis. However, such
increases might also occur if protein synthesis were
blocked or slowed down. In the nitrate-N treatments in-
creases in soluble amino nitrogen did not occur, which
may indicate that blockage of protein synthesis was more
important in the high temperature effects found than was
protein hydrolysis.

The results of the soil experiments also indicated
that a blockage or slow down of protein synthesis was
more likely to be involved than an increase in protein
hydrolysis. In Experiment 2 when no nitrogen was added,
the protein nitrogen content was lower at the low tempera-
ture than at the high temperature. When nitrogen was
added, no differences due to temperature occurred. Fol-
lowing the first harvest, as growth continued at the low
temperature, protein nitrogen decreased for all treat-
ments. At the high temeprature, as a small amount of
regrowth occurred in the tops, protein nitrogen dropped.
However, in the roots where no new growth occurred, pro-
tein nitrogen remained the same. Thus, the lower protein

nitrogen at the low temperature when no nitrogen was added

 

.57

and the decreases in protein nitrogen between harvests at
the low temperature seem to indicate that as growth con-
tinued and as available nitrogen became low, as probably
occurred in the soil experiments, protein was broken down
in the older portions of the plants and resynthesized for
new growth. The decreases in protein content Observed
were not likely to be simply dilutions with growth since
new growth is generally high in protein content. On the
other hand, at the high temperature little or no growth
was occurring, possibly because Of a lack of protein syn-
thesis, so no breakdown and resynthesis of protein occurred.
If protein synthesis were blocked one would also
expect some increase in soluble amino nitrogen as these
precursors to protein built up. If available nitrogen
were low, the build up would not be expected to be as
great. On the other hand, if a rapid hydrolysis Of pro-
tein without resynthesis were occurring, one would expect
the soluble amino nitrogen to remain high even if avail-
able nitrogen dropped. The changes in soluble amino
nitrogen observed in the soil experiments followed the
former pattern. The soluble amino nitrogen content
reached quite low levels especially in Experiment 3. It
also decreased between harvests in Experiment 2. Although
the soluble amino nitrogen content remained higher at
the high temperature compared to the low temperature,

the proportion Of soluble amino nitrogen to protein

 

58

nitrogen actually decreased between harvests at the high
temperature, a result that would not be expected if a
general breakdown of protein were occurring.

Increases in free ammonium and amide nitrogen are
also generally associated with protein breakdown, but
these fractions remained low at the high temperature,
although they did respond to increased nitrogen levels.

It is possible that some injury could occur from
an accumulation of some particular soluble nitrogen
fraction, e.g. free ammonia (11, 1A). No particular
soluble nitrogen compound accumulated to high levels in
the soil experiments. In the nutrient solution experi—
ment the soluble nitrogen fraction was much higher, but
the constituents of this fraction were not determined.
Thus, it is possible that a particular toxic compound
might accumulate at higher nitrogen levels. This could
also happen at higher temperature conditions such as used
by Petinov and Molotkovskii (21, 22).

The interactions of nitrogen source and level and
temperature on growth and on the chemical composition of
cool-season grasses indicate that these variables must
be carefully controlled in future studies of the bio-

chemical basis for high temperature growth reduction.

CONCLUSIONS

1. An interaction between nitrogen nutrition and
temperature on the growth of cool—season grasses occurred.
In a nutrient solution experiment, where the nitrogen
levels remained constant throughout the experimental
period, growth was always reduced by the high tempera-
ture, and the reduction was greater at high levels Of
nitrogen and with ammonium-N. At the low temperature an
increase in level of nitrate-N increased growth while an
increase in level of ammonium-N decreased growth. In a
soil experiment in which nitrification of added ammonium-N
was not prevented, additions of either ammonium or
nitrate-N caused an increase in growth of tops at the low
temperature but not at the high temperature. In a second
soil experiment in which nitrification of added ammonium-N
was prevented, ammonium—N was superior to nitrate-N for
tOp growth. An increase in growth for the high level of
ammonium—N but not for the high level of nitrate—N occurred
at the low temperature. No response to source or level of
nitrogen occurred at the high temperature.

2. Changes in soluble carbohydrate content did not
appear to be the causal factor of growth reduction at the

high temperature nor for the interactions found. In no case

59

 

IT

60

was soluble carbohydrate exhausted, nor present in low
enough concentrations to limit growth.

3. In the nutrient solution experiment protein
nitrogen content appeared to be slightly increased by
ammonium—N compared to nitrate—N and slightly decreased
by high temperature. The soluble amino nitrogen content .
generally was higher with ammonium—N than nitrate—N and 3
increased at the high temperature with ammonium—N but not

with nitrate—N.

 

A. In the first soil experiment protein nitrogen
content was generally lower at the low temperature than
at the high temperature with no added nitrogen. When
nitrogen was added, no differences due to temperature
occurred at the first harvest. Between harvests protein
nitrogen content decreased where growth continued but
remained constant in the roots at the high temperature
where no growth occurred. The soluble amino nitrogen con-
tent was generally higher with nitrogen additions and at
the high temperature. The level of available nitrogen
appeared to exert a greater effect than did temperature.
Between harvests the soluble amino nitrogen content
decreased at both temperatures. In the second soil exper-
iment where free ammonium, nitrate, glutamine, and aspara-
gine nitrogen were determined, these fractions all were
quite low and generally responded to an increase in

ammonium nitrogen level but not to temperature.

61

5. The effects of nitrogen nutrition and temperature
on nitrogen metabolism appeared more likely to involve a
blockage or slow down of protein synthesis rather than an
increase in protein breakdown. An accumulation of some
toxic nitrogen compound at high nitrogen levels could also

be involved.

 

BIBLIOGRAPHY

62

BIBLIOGRAPHY

ALTERGOTT, V. F. 1936. The cause of death Of plants
at high temperatures. Izv. Akad. Nauk. S.S.R.
Biol. Ser. 1:79—88. Abst. in Herb. Abst.
7:Al—A2, 1937.

BEARD, J. B. and DANIEL, W. H. 1967. Variations in
the total, nonprotein, and amide fractions of
Agrostis palustris Huds. leaves in relation to
certain environmental factors. Crop. Sci.
7:111—115.

 

BEINHART, G. 1963. Effects of environment on
meristematic development, leaf area, and growth
of white clover. Crop. Sci. 3:209-213.

BOMMER, D. F. R. 1966. Influence Of cutting fre—
quency and nitrogen level on the carbohydrate
reserves of three grass species. Proc. X Int.
Grassl. Cong. 156-160.

BROWN, E. M. 19A3. Seasonal variations in the growth
and chemical composition of Kentucky bluegrass.
Missouri Ag. Exp. Sta. Res. Bull. 360. pp 1—56.

BROYER, T. C. and HOAGLAND, D. R. l9A0. Methods of
sap expression from plant tissues with special
reference to studies on salt accumulation by
excised barley roots. Am. J. Bot. 27:501—511.

DARROW, R. A. l9A0. Effects of soil temperature,
pH, and nitrogen nutrition on the development
of Poa pratensis. Bot. Gaz. 101:109-127.

 

DUFF, D. T. 1967. Some effects of supraoptimal
temperatures upon creeping bentgrass (Agrostis
palustris Huds.). Ph.D. thesis. Michigan State
University. pp 1—61.

 

GORING, C. A. I. 1962. Control of nitrification by
2-chloro-6—(trichloromethyl) pyridine. Soil
Sci. 93:211—218.

10.

11.

12.

13.

1A.

15.

16.

17.

18.

19.

20.

6A

GREEN, D. G. 1963. Seasonal responses of soluble
carbohydrates in the leaves of four cool—season
grasses to five nitrogen treatments. M.S.
thesis. Michigan State University. pp 1-53.

GREENFIELD, S. S. 19A2. Inhibitory effects of
inorganic compounds on photosynthesis in
Chlorella. Am. J. Bot. 29:121—131.

 

HARRISON, C. M. 193A. Response of Kentucky blue—
grass to variations in temperature, light,
cutting, and fertilizing. Plant Physiol.
9:83-106.

HODGE, J.E. AND HOFREITER, B. T. 1962. Determina-
tion of reducing sugars and carbohydrates. In:
Methods in Carbohydrate Chemistry Vol. I. R. L.
Whistler and M. L. Wolfrom, Eds. Academic Press,
Inc. pp 380-39A.

KROGMAN, D. W., JAGENDORF, A. T., and AVRON, M. 1959.
Uncouplers of spinach chloroplast photosynthetic
phosphorylation. Plant Physiol. 3A:272-277.

LANGRIDGE, J. 1963. Biochemical aspects of tempera-
ture response. Annual Rev. Plant Physiol.

lAzAAl-A62.

LAWRENCE, J. M. and GRANT, D. R. 1963. Nitrogen
mobilization in pea seedlings. II. Free amino
acids. Plant Physiol. 38:561—566.

LEITCH, I. 1916. Some experiments on the influence
of temperature on the rate of growth of Pisum
sativum. Ann. Bot. 30:25-A6.

LEVITT, J. 1956. The Hardiness of Plants. Academic
Press, Inc. pp 1—278.

MITCHELL, K. J. 1956. Growth of pasture species
under controlled environment. 1. Growth at
various levels of constant temperature. New
Zealand J. Sci. Technol. A 38:203-216.

PELLETT, H. M. and ROBERTS, E. C. 1963. Effects
of mineral nutrition on high temperature induced
growth retardation of Kentucky bluegrass.
Agron. J. 55:A73—A76.

21.

22.

23.

2A.

25.

26.

27.

28.

29.

30.

65

PETINOV, N. S. and MOLOTKOVSKII, Yu. G. 1951.
[Protective reactions in heat—resistant plants
induced by high temperatures.] Fiziologiya
Rastenii A:225-228. A.I.B.S. Translation Soviet
Plant Physiol. A:221—228. 1957.

PETINOV, N. S. and MOLOTKOVSKII, Yu. G. 1960. [The
effect of respiratory inhibitors on heat resis-
tance in plants.] Fiziologiya Rastenii
7:665-672. A.I.B.S. Translation Soviet Plant

Physiol. 7:551-556. 1960.
ROSEN, H. 1957. A modified ninhydrin colorimetric T_
analysis for amino acids. Arch. Biochem. '“

Biophys. 67:10-15. '

 

 

SAYRE, J. D. and MORRIS, V. H. 1932. Use of expres-
sed sap in determining the composition of corn
tissue. Plant Physiol. 7:261-272.

SPRAGUE, H. B. 193A. Utilization of nutrients by
colonial bent (Agrostis tenuis) and Kentucky
bluegrass (Poa pratensis). New Jersey Ag. Exp.
Sta. Bull. 570. pp 1-16.

 

 

STEWARD, F. C. and STREET, H. E. 19A6. The soluble
nitrogen fractions of potato tubers; the amides.
Plant Physiol. 21:155-193.

STEWARD, F. C. 1963. Effects of environment on
metabolic patterns. In: Environmental Control
of Plant Growth. L. T. Evans, Ed. Academic
Press, Inc. pp l95—21A.

 

SULLIVAN, J. T. and SPRAGUE, V. G. 19A9. The effect
of temperature on the growth and chemical compo—
sition of the stubble and roots of perennial
ryegrass. Plant Physiol. 2A:706—719.

SULLIVAN, J. T. and SPRAGUE, V. G. 1953. Reserve
carbohydrates in orchard grass cut for hay.
Plant Physiol. 28:30A—3l3.

SYRETT, P. J. 1956. The assimilation of ammonia
and nitrate by nitrogen—starved cells of
Chlorella vulgaris. II. The assimilation of
large quantities of nitrogen. Physiol.
Plantarum 9:19-27.

 

31.

32.

33.

3A.

35.

66

SYRETT, P. J. 1956. The assimilation of ammonia and
nitrate by nitrogen-starved cells of Chlorella
vulgaris. III Difference of metabolism dependent
on the nature of the nitrogen source. Physiol.
Plantarum 9:28-37.

VARNER, J. E., BULEN, W. A., VANECKO, S., and BURRELL,
R. C. 1953. Determination of ammonium, amide,
nitrite, and nitrate nitrogen in plant extracts.
Anal. Chem. 25:1528-1529.

VICKERY, H. B., PUCHER, G. W., CLARK, K. E., CHIBNALL,
A. C., and WESTFALL, R. G. 1935. The determina—
tion of glutamine in the presence of asparagine.
Biochem. J. 29:2710-2720.

WHITE, P. R. 1932. Influence of some environmental
conditions on the growth of excised root tips of
wheat seedlings in liquid media. Plant physiol.
7:613-628.

WHITE, P. R. 1937. Seasonal flucuations in growth
rates of excised tomato root tips. Plant
Physiol. 12:183—190.

.IIIIIIIJIIIIIIII

3

 

“ll

WI
TM
5"
NM
mIWIIH
Wm