68-8312 RAUSCHKOLB, Roy S., 1933- NTTROGEN ABSORPTION AND UTILIZATION BY

68-8312 RAUSCHKOLB,  Roy S., 1933- NTTROGEN ABSORPTION AND  UTILIZATION BY
This dissertation has been
microfilmed exactly as received
68-8312
RAUSCHKOLB, Roy S., 1933NTTROGEN ABSORPTION AND UTILIZATION BY
GOSSYPIUM HIRSUTUM AS INFLUENCED BY
NITROGEN SOURCE.
University of Arizona, Ph.D.f 1968
Agriculture, soil science
University Microfilms, Inc., Ann Arbor, Michigan
NITROGEN ABSORPTION AND UTILIZATION BY GOSSYPIUM HIRSUTUM
AS INFLUENCED BY NITROGEN SOURCE
by
Roy S. Rauschkolb
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF AGRICULTURAL CHEMISTRY AND SOILS
In Partial Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
1 9 6 8
THE UNIVERSITY OF ARIZONA
GRADUATE COLLEGE
I hereby recommend that this dissertation prepared under my
direction by
entitled __
Roy S. Rmmehkolb
£_
Ml trow Abtorptlon and Utill—tlon far
»• laflmncid far litrogen sbmm.
be accepted as fulfilling the dissertation requirement of the
degree of
Doctor of PMlmophy .
y
Dissertation Director
, 3A /# 7
Dace
After inspection of the dissertation, the following members
of the Final Examination Committee concur in its approval and
recommend its acceptance:*
Y -<h //u'^
5^
-a
Jq,-/
*This approval and acceptance is contingent on the candidate's
adequate performance and defense of this dissertation at the
final oral examination. The inclusion of this sheet bound into
the library copy of the dissertation is evidence of satisfactory
performance at the final examination.
STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of
requirements for an advanced degree at The University of Arizona and is
deposited in the University Library to be made available to borrowers
under rules of the Library.
Brief quotations from this dissertation are allowable without
special permission, provided that accurate acknowledgment of source is
made. Requests for permission for extended quotation from or reproduc­
tion of this manuscript in whole or in part may be granted by the head
of the major department or the Dean of the Graduate College when in his
judgment the proposed use of the material is in the interests of scholar­
ship. In all other instances, however, permission must be obtained from
the author.
ACKNOWLEDGMENT
The author gratefully acknowledges the assistance of numerous
people who have contributed to the preparation of this dissertation both
in a physical sense and in spirit.
Special appreciation is extended to Dr. T. C. Tucker for his
guidance and assistance over the years.
The author also wishes to acknowledge the technical aid which
was extended him by Silver Darner and Richard Dupont.
A most sincere thank you is extended to the author's wife, Joan,
whose personal sacrifice, perserverance, understanding, and affection
has donated immeasurably to this achievement.
iii
TABLE OF CONTENTS
ge
LIST OF TABLES
vi
LIST OF FIGURES
ii
ABSTRACT
ix
INTRODUCTION
1
LITERATURE REVIEW
3
Absorption Studies ...
3
Metabolism of Plant Nitrogen Fractions
9
15
METHODS
15
Procedure for Growing Plants
Short Term Nitrogen Absorption Studies . . . .
16
Deficiency Study ...
19
Chemical Analyses
20
RESULTS AND DISCUSSION
21
Preliminary Growth Chamber Studies
,
21
29
Effect of Light Regime on Absorption
Effect of Light Regime on Utilization
....
31
Light-Dark Cycle
32
Prolonged Light
41
Prolonged Dark
49
Effect of Nitrogen Deficiency on Absorption and
Utilization
58
Absorption
58
Utilization
58
iv
V
Page
SUMMARY AND CONCLUSIONS
73
APPENDIX
74
REFERENCES
80
LIST OF TABLES
Table
1.
Page
THE AMOUNT OF NITROGEN IN VARIOUS FRACTIONS
OF TOPS AND ROOTS AS INFLUENCED BY
ORGANIC N-SOURCES
27
CONCENTRATION OF AMMONIUM AND NITRATE IN THE
CULTURE SOLUTION CONTAINING ORGANIC
N-SOURCES
28
3.
NITROGEN ABSORBED FRCM ORGANIC N-SOURCES
28
4.
ABSORPTION OF NITROGEN FROM DIFFERENT N-SOURCES
IN NUTRIENT SOLUTION AS INFLUENCED BY
LIGHT REGIME
30
THE EFFECT OF NITROGEN SOURCE AND DEFICIENCY
ON ABSORPTION
59
PER CENT TOTAL NITROGEN IN DRY PLANT TISSUE—
TOPS AND ROOTS
69
MEANS OF MAIN EFFECTS EXPRESSED AS PER CENT TOTAL
NITROGEN IN DRY PLANT TISSUE—TOPS AND ROOTS
70
PRELIMINARY GROWTH CHAMBER STUDY WITH AMMONIUM
AND NITRATE N-SOURCES—TOPS AND ROOTS
75
THE NITROGEN FRACTIONS OF PLANTS GROWN IN THE
LIGHT-DARK CYCLE WITH VARIOUS INORGANIC
N-SOURCES—TOPS AND ROOTS
76
THE NITROGEN FRACTION OF PLANTS GROWN IN THE
PROLONGED LIGHT CYCLE WITH VARIOUS INORGANIC
N-SOURCES —TOPS AND ROOTS
77
THE NITROGEN FRACTION OF PLANTS GROWN IN THE
PROLONGED DARK CYCLE WITH VARIOUS INORGANIC
N-SOURCES—TOPS AND ROOTS
78
THE NITROGEN FRACTION OF PLANTS GROWN WITH
INORGANIC N-SOURCES FOR DIFFERENT NITROGEN
REGIMES—TOPS AND ROOTS
79
2.
5.
6.
7.
8.
9.
10.
11.
12.
vi
LIST OF FIGURES
Figure
Page
1.
TOTAL KJELDAHL-N IN PLANT TISSUE—TOPS AND ROOTS
22
2.
AMMONIUM, NITRATE, AND TOTAL ALCOHOL-SOLUBLE
REDUCED NITROGEN FRACTIONS—TOPS AND ROOTS
23
AMMONIUM FRACTION OF LIGHT-DARK CYCLE PLANTS—
TOPS AND ROOTS
33
NITRATE FRACTION FOR LIGHT-DARK CYCLE PLANTS—
TOPS AND ROOTS
34
TOTAL ALCOHOL-SOLUBLE REDUCED-N FOR LIGHT-DARK
CYCLE PLANTS—TOPS AND ROOTS . . . .
. 35
3.
4.
5.
. 6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
TOTAL KJELDAHL-N IN DRY PLANT TISSUE FROM
LIGHT-DARK CYCLE PLANTS—TOPS
36
TOTAL KJELDAHL-N IN DRY PLANT TISSUE FROM
LIGHT-DARK CYCLE PLANTS—ROOTS
37
AMMONIUM FRACTION OF LIGHT CYCLE PLANTS—
TOPS AND ROOTS
42
NITRATE FRACTION OF LIGHT CYCLE PLANTS —
TOPS AND ROOTS
44
TOTAL ALCOHOL-SOLUBLE REDUCED-N OF LIGHT CYCLE
PLANTS—TOPS AND ROOTS
45
TOTAL KJELDAHL-N IN DRY PLANT TISSUE OF LIGHT
CYCLE PLANTS—TOPS
47
TOTAL KJELDAHL-N IN DRY PLANT TISSUE OF LIGHT
CYCLE PLANTS—ROOTS
48
AMMONIUM FRACTION OF DARK CYCLE PLANTS—
TOPS AND ROOTS
:..... 50
NITRATE FRACTION OF DARK CYCLE PLANTSTOPS AND ROOTS
52
TOTAL ALCOHOL-SOLUBLE REDUCED-N OF DARK CYCLE
PLANTS—TOPS AND ROOTS
54
vii
viii
Figure
16.
17.
Page
TOTAL KJELDAHL-N IN DRY PLANT TISSUE OF DARK
CYCLE PLANTS—TOPS
55
TOTAL KJELDAHL-N IN DRY PLANT TISSUE OF DARK
CYCLE PLANTS—ROOTS
57
AMMONIUM FRACTION FOR DIFFERENT NITROGEN REGIMES
AND SOURCES—TOPS AND ROOTS
60
NITRATE FRACTION FOR DIFFERENT NITROGEN REGIMES
AND SOURCES—TOPS AND ROOTS
'
62
TOTAL ALCOHOL-SOLUBLE REDUCED-N FRACTION FOR
DIFFERENT NITROGEN REGIMES AND SOURCES—
TOPS AND ROOTS
64
TOTAL KJELDAHL-N FRACTION FOR DIFFERENT NITROGEN
REGIMES AND N-SOURCES—TOPS
66
TOTAL KJELDAHL-N FRACTION FOR DIFFERENT NITROGEN
REGIMES AND N-SOURCES—ROOTS
67
7
18.
19.
20.
21.
22.
ABSTRACT
The absorption and utilization of various forms of nitrogen by
young cotton plants (Gossypium hirsutum L. var. Acala 4-42) was studied
using a short term absorption period.
Both organic and inorganic nitro­
gen sources were supplied to the plants which were grown in culture solu
tions.
Most of the emphasis was on the effect of light and degree of
nitrogen deficiency on the assimilation of inorganic forms of nitrogen.
The relative changes in various nitrogen fractions were observed in the
tops and roots of plants grown in both the greenhouse and growth chamber
The absorption and utilization of nitrogen appeared to be de­
pendent upon both the presence or absence of light and the nitrogen
source.
Nitrogen assimilation was affected the most by light or dark
when ammonium was the only source of nitrogen.
The degree of nitrogen deficiency was found to have a marked in­
fluence on the absorption and utilization of the various inorganic nitro
gen sources.
The most dramatic effect occurred in cotton plants which
were extremely nitrogen deficient.
In these plants, large increases
occurred in each of the fractions studied except the ammonium fraction
when nitrate was the sole source of nitrogen. This was a clear cut
demonstration of a preference for a specific form of nitrogen by cotton
plants under these environmental conditions.
ix
INTRODUCTION
The eclectic ability of Gossypium hirsutum, with respect to
nitrogen, has long been studied.
The response obtained for various
N-sources has depended upon environmental factors and age of the plants.
However, for any given environmental variable, different responses for
different N-sources may be elicited.
To determine the effectiveness of different N-sources to supply
nitrogen to cotton plants, three approaches have been employed:
(1) meas­
ure the disappearance of nitrogen from culture solutions containing dif­
ferent N-sources, (2) follow the nitrogen concentration in different
nitrogen pools in various parts of the plant, and (3) use yield in field
studies as a measure of the utilization of different N-sources by cotton
plants.
The last has been an ineffective method for determining whether
cotton plants are selective with regard to N-source.
The first two approaches have been studied individually and oc­
casionally in conjunction with one another.
When studied together,
selected plant parts have been used to evaluate the utilization of the
various N-sources; the roots have been nefariously neglected in these
studies.
Diurnal changes as they influence the nitrogen pools in the plant
have been investigated using aerial portions of the plant exclusively.
In addition, studies involving the absorption of various N-sources and
the relative changes within the roots and the tops of plants to diurnal
changes are non-existant.
1
2
The influence of nitrogen deficiency on the assimilation of
nitrogen has been studied in various plants.
However, a perusal of the
literature revealed a paucity of studies evaluating the effect of nitro­
gen deficiency on the absorption and utilization of different nitrogen
sources in cotton plants.
The objectives of these studies were to determine if cotton
plants would exhibit a preference for any of the N-sources used in the
experiments when supplied in culture solutions, and to determine if
light or degree of nitrogen deficiency were some of the conditions under .
which a preference for an N-source could be demonstrated.
The nitrogen
levels in both the tops and roots were determined to evaluate the rela­
tive changes within them with respect to the conditions imposed upon the
plants.
A corollary of the study was the evaluation of short term ab­
sorption periods as an effective means for studying the assimilation of
nitrogen.
LITERATURE REVIEW
The underlying theme of all the research conducted on cotton
(Gossypium hirsutum) with various forms of nitrogen has been that some
form of nitrogen was preferentially absorbed.
The major factor contrib­
uting to this premise was the often observed, but little documented,
response in the field of a rather quick recovery of nitrogen deficient
plants when nitrate nitrogen (nitrate-N) was supplied the plants as com­
pared to applications of other nitrogen sources (N-sources).
This has
been at least partially documented by Gardner (6), who found that the
nitrate level in cotton petioles was indicative of the nitrate level in
the soil.
Additions of ammonium nitrate to nitrogen deficient plants
caused the nitrate level in the petioles to increase.
More commonly,
what has been observed is that when nitrogen has been supplied initially
in various forms there has been little difference in yield (1, 8, 31);
however, some investigators have found urea (17, 38), ammonium (32), and
nitrate (18), each have produced the highest yields of cotton when sup­
plied individually.
These experiments reveal very little as to the
plant"s preference for a specific form of nitrogen because of the con­
version of N-sources by soil microorganisms.
Absorption Studies
The ability of organic N-sources to supply nitrogen for several
plants has been investigated.
Hattori (9) found that nitrogen starved
cells of Chlorella ellipoidea assimilated nitrogenous compounds in the
3
4
following order:
citrulline.
ammonia, arginine, urea, nitrate, ornithine, and
Ghosh and Burris (7) using tomato (Lycopersicum esculentum),
tobacco (Nicotiana tabacum), algae (Chlorella pyrenoidosa), and clover
(Trifolium pratense), found that when some ^-amino acids were supplied
individually, they were capable of supporting plant growth.
When the
-amino acids which caused inhibition of growth were used in combination
with the others no such inhibition was observed.
Virtanen and Linkola (34) found aspartic acid was an effective
N-source for peas (Pisum sativum).
They also found it was absorbed
simultaneously with ammonium and nitrate forms of nitrogen.
The absorption of urea by tomatoes (Lycopersicum esculentum) has
been studied by Kirby and Mengel (16).
After growing 14 days in a culture
solution, plants supplied with nitrate-N produced the greatest weight and
plants supplied with ammonium-N the least.
were intermediate in weight.
The plants supplied with urea
A perusal of the literature indicates there
have been very few, if any, absorption studies conducted on cotton using
organic sources of nitrogen.
Most of the information available concerns
absorption of the inorganic forms of nitrogen prevalent in soils, namely,
ammonium and nitrate.
The absorption studies conducted with ammonium and nitrate forms
of nitrogen have been concerned not only with the efficacy of these two
sources, individually, but with the influence of pH, aeration, and the
cation-anion balance of the culture solution on absorption. The effect
of one source of nitrogen on the absorption of the other has also been
investigated.
5
When ammonium and nitrate were used alone and in various concen­
tration combinations Naftel (22) found that the amount of either source
absorbed depended on the age of the cotton plants.
were 24 and 48 hours.
Absorption periods
The younger plants absorbed more ammonium-N while
the older plants absorbed more nitrate-N from the culture solution.
Four-week-old plants absorbed approximately equal amounts from either
source.
However, more total nitrogen was absorbed from culture solutions
by four-week-old plants when both N-sources were present than when either
N-source was present alone.
Holley and Dulin (13) found that varying the concentration of
nitrate in the culture solution had very little effect on the nitrate
concentration in the sap or on the per cent total nitrogen in the dry
matter of cotton plants.
The nitrogen concentration in the solutions
ranged from 2.8 to 12.0 meq/1.
As the plants grew older and absorbed
more nitrogen from the solution, increases in the nitrate concentration
of the solution of up to 6 times the low value did not change the per
cent total nitrogen in the dry matter.
When ammonium-N was used, how­
ever, they found that as the nitrogen concentration increased, the
nitrogen content of the various fractions in the sap and the per cent
nitrogen in the dry matter increased.
They attributed the difference in
response to variations in the concentration of the two N-sources to the
fact that nitrate is accumulated in the tissue of the plants whereas
ammonium is not.
It is generally agreed that most plants reach their optimum
growth in the pH range of 6.0 to 8.0. It is also equally well known
6
that plants can, and do, grow at pH values outside that range.
Much at­
tention has been given to the effect of pH on the absorption and utiliza­
tion of ammonium and nitrate nitrogen.
Paden and Garman (24) found that yields of seed cotton from soil
plots at different pH levels increased as the pH value of the soil in­
creased from 5.0 to 6.5.
In an experiment conducted by Naftel (22) the
effect of different pH levels on the absorption of ammonium and nitrate
ions from a culture solution was observed.
He found that varying the
pH from 3.5 to 7.0 had little effect on the absorption of nitrate-N. The
absorption of ammonium-N increased as the pH of the culture solution in­
creased.
The greatest amount of total nitrogen absorbed usually occurred
at pH 6.0.
In another study, Barker et al. (2) using bean plants (Phaseolus
vulgaris) found no difference due to the control or non-control of acid­
ity on the absorption of ammonium from the culture medium for a period
of 2 days.
However, after 2 days, controlling the pH of the medium
caused an increase of the absorption of ammonium-N.
Lycklama (19) found that the absorption of ammonium from a cul­
ture solution by perennial rye grass (Lolium perenne) varied only slightly
in the pH range 4.0 to 6.5.
Also, when sulfate was the accompanying
anion, there was only a very slight decrease in the absorption of
ammonium as the pH increased from 7.0 to 8.5.
optimum pH for nitrate absorption was 6.2.
He also found that the
Separate studies conducted
by Dejaegere (5) and Ivanova (15) indicated that optimum growth of cotton
was obtained with pH values in the alkaline range.
These tests were
conducted in potted soils and culture solutions, respectively.
7
The effect of pH on the absorption and utilization of ammonium-N
and nitrate-N differs with respect to the length of time the plants are
exposed to the growth medium.
In studies conducted where the exposure
time to the culture solution has been for a range of time of a few min­
utes to 3 days, the effect of pH on the absorption of ammonium and nitrate
ions was slight; also, the absorption of either N-source seems to be en­
hanced in the pH range 4.0 to 6.0.
Where studies have been conducted
with exposure times of several weeks at different pH levels, the plants
grew best in the pH range of 6.5 to 8.0.
Another factor which influences the absorption of nitrogen is
the concentration of other cations and anions in the culture solution.
The effect of anion to cation ratio on the growth of cotton was studied
by Neirinckx (23).
He varied the anion (nitrate, sulfate, and phosphate)
to cation (potassium, calcium, and magnesium) ratio from 0.75 to 2.0.
He found that a maximum production of plant material occurred at an
anion to cation ratio of 1.33.
Dejaegere (5) showed that optimal dry
matter production was obtained in the pH range of 6.8 to 8.5 when low
nitrate to ammonium ratios corresponded to high anion to cation ratios.
These investigators have all concluded that the concentration of the
element in the plant is dependent upon the concentration in the nutrient
supply.
The concentration of an element in the plant varies with its
concentration in the medium; although the amount of variation in the
plant is not as great as in the medium.
This conclusion is not in agree­
ment with the result found by Holly and Dulin (13) with respect to nitro­
gen from various sources.
8
Aeration has been found by Pershin (25) to have an effect on the
absorption of ammonium-N and nitrate-N by cotton from a culture solution.
The effect of aeration varied with the concentration of nitrogen in the
solution.
At the lower nitrogen concentration value (6 mgm/1) absorption
of ammonium from the culture solution with aeration after 24 hours was
greater than the absorption of ammonium without aeration, and greater
than nitrate absorption either with or without aeration.
However, the
sum of the nitrogen absorbed from both N-sources was slightly higher
from the aerated solution.
At the higher concentration value (10 mgm/1),
the absorption of nitrate was greater than the absorption of ammonium
with aeration.
The absorption of ammonium without aeration was much
lower than any of the other values.
At this concentration level, the
sum of nitrogen absorbed from both N-sources was much higher with aera­
tion than without aeration.
Other environmental factors that could influence the absorption
of a m m o n i u m - N a n d n i t r a t e - N a r e t e m p e r a t u r e a n d d e g r e e o f n i t r o g e n d e ­
ficiency.
In studying the assimilation of ammonium and nitrate by
nitrogen-starved cells of Chlorella vulgaris, it was found by Syrett (30)
that initially ammonium was more quickly absorbed from the medium than
nitrate.
At the termination of the experiment (150 minutes) the rates
were the same, although the amount of nitrogen absorbed as ammonium was
much greater than the amount absorbed as nitrate.
The effect of changes in temperature on the absorption of ammon­
ium and nitrate was studied by Lycklama (19) with perennial rye grass
seedlings.
He found that as the temperature increased from 5° C to ap­
proximately 30° C there was an increase in the absorption of both
; nnonium and nitrate.
As the temperature increased beyond 30° C, tho
absorption of ammonium decreased and the absorption of nitrate continued
to increase.
Lycklama has also studied the effect of varying concentrations
of one source on the absorption of the other.
He found that as the
nitrate concentration of the culture solution varied, the absorption
rate of ammonium decreased slightly.
The maximum nitrate concentration
of the solution was 3 meq/1, and the ammonium concentration was held
constant at 0.2 meq/1.
However, when the ammonium concentration in a
solution was increased to a maximum of 0.9 meq/1, while the nitrate
concentration was held constant at 1.0 meq/1, there was a drastic reduc­
tion in the nitrate absorption.
It appears that as the ammonium concen­
tration increases, the nitrate absorption rate approaches some minumum
value.
On the other hand, Naftel (22) in his studies with cotton plants
found the age of the plants had more to do with the absorption of an
N-source than the effect of one N-source on the other.
The total amount
of nitrogen absorbed was greater where the two N-sources were present
until the plants reached 11 weeks of age at which time the absorption of
nitrogen from the nitrate N-source was greater.
Metabolism of Plant Nitrogen Fractions
Practically every form of nitrogen in the plant has been studied
in conjunction with the overall nitrogen nutrition of the plant end the
absorption of nitrogen from the soil or culture solutions.
This is not
to say that each different nitrogen-containing compound in the plant has
been investigated.
In general, the nitrogen in the plant has been broken
10
down into the following categories: ammonium, nitrate, anide, 0(-£i»inc
acid, soluble (either present in the sap or alcohol soluble), alcohol
insoluble, and total Kjeldahl-N.
nitrates.
The latter may or may not include
The insoluble fraction is sometimes referred to as the pro­
tein fraction.
This fraction is obtained by subtracting the total
soluble reduced-N from the total Kjeldahl-N determined on the dry plant
tissue.
The total soluble reduced-N, qualitatively, contains some very
important metabolic intermediates; however, other than
-amino acids
and amides, no attempt has been made to delineate them.
Quantitatively,
each individual compound contributes only a small portion to the total.
Also, if one assumes a dynamic equilibrium within the plant, then changes
in the nutritional status of the plant will be reflected in the nitrogen
level of the "metabolic nitrogen pool."
Holley et al_. (12) studied the effect of ammonium and nitrate
sources on the amide and ^-amino acid content of sap expressed from
cotton plants.
They showed that when ammonium was the N-source the
nitrogen content in these fractions was higher than when nitrate was the
N-source.
They concluded from this that nitrate was converted to organic
forms of nitrogen in the plant more slowly than ammonium.
Their conclu­
sion that nitrification under field conditions was undesirable is believed
to be somewhat premature.
One could just as easily conclude from their
data that ammonium was in some manner inhibiting the utilization of the
forms studied, and therefore these forms accumulated with ammonium as the
N-source, or that an inhibition of nitrate reduction occurred.
It also
11
is possible that nitrate reduction proceeded at an orderly rate approxi­
mately equally to the rate of conversion of amides and amino acids to
protein.
Vines and Wedding (35) in studying garden beet (Beta vulgaris)
mitochondria concluded that ammonia, either as a salt or dissolved
gaseous form, caused an inhibition of dihydronicotinamide adenine
dinucleotide (NADH) oxidation at high pH values.
The basis for their
conclusion was the fact that the metabolism of the Krebs cycle interme­
diates was disrupted except for succinic acid.
Succinic acid reduction
is known to proceed without the presence of NADH.
tend to corroborate this finding.
Kirby and Mengel (16)
They found that when tomato plants
were grown in culture solutions with urea, ammonium,and nitrate as dif­
ferent N-sources, the non-volatile organic acid content of all the plant
parts studied was greater with nitrate as the sole N-source.
Lycklama (19) studied the effects of the presence of both ammonium
and nitrate on the accumulation and reduction of nitrate with excised
roots of perennial rye grass.
He found that the accumulation of nitrate
in the roots was not affected by the presence of ammonium in the culture
solution.
Moreover, he found that in the presence of ammonium the reduc­
tion of nitrate was curtailed.
From these data he concluded that the
accumulation and reduction of nitrate are two different and distinct
processes.
The assimilation of ammonium and nitrate was studied by Syrett
(30).
He indicated that the conversion rate of ammonium to
-amino
acids was faster than the conversion rate from ^C-amino acids to protein.
12
With nitrate there was no difference in the rates.
He also concluded
that the reason nitrate was not assimilated as readily as ammonium was
because the respiration of polysaccharides was competing with nitrate
for NADH.
He proposed the following schematic diagram to describe the
pathway of nitrogen assimilation.
-—
Polysacch
'"-
v
Soluble
Insoluble
Organic-N—^Organic-N
0
2
Weissman (37) studied the effect of ammonium and nitrate nutrition on the protein level and exudate of sunflower plant (Helianthus
annuus) leaves.
He showed that when ammonium was the N-source there was
no nitrate present in the leaf exudate.
With ammonium and nitrate in
combination, or with nitrate along", there was nitrate present in the
exudate and the level of the ammonium fraction was higher than with the
ammonium N-source alone.
The amide fraction of the exudate was greatest
with ammonium alone and least with nitrate alone.
In studying the pro­
tein level of older and younger leaves, he found that in either case the
level was greatest when ammonium and nitrate were used in combination.
The total nitrogen was also determined on the leaves using the Kjeldahl
method modified to include nitrates.
In this instance, the total nitro­
gen was greater in the younger leaves with the higher level in the leaves
of plants grown with nitrate as the only N-source.
13
Vomhof (36) studied several nitrogen fractions with respect to
different plant parts of cotton at different stages of growth. He found
that approximately one-third of the total soluble reduced-N is made up
of 0^-amino acids at the 5 leaf stage of growth.
He also found a high
degree of correlation between the nitrogen pools within the soluble
fraction and with the soluble total.
In a series of papers by Phillis and Mason (26, 27) and Maskell
and Mason (20, 21), the diurnal fluctuations of the nitrogen content of
expressed sap and dried tissue from cotton plants was studied. Several
nitrogen fractions were studied in various plant parts. They showed
that the protein level in the leaves was markedly influenced by light
and dark periods.
Furthermore, the bark and wood contained approximately
equal amounts of nitrogen and there was no appreciable diurnal variation
in this part of the plant. They also found that the upper portions of
the plant parts studied contained more of each of the fractions studied
than the lower portions. These latter results are in agreement with the
results found by Vomhof (36).
The preceding literature review has been concerned principally
with the two areas of investigation; the effect of environment on the
absorption of nitrogen in various forms and the utilization of these
various N-sources. These studies have been conducted on several plants,
including cotton.
In cotton plants the studies were more concerned with
either absorption or utilization and seldom with the combination of the
two.
14
The study undertaken herein, endeavored to evaluate the absorp­
tion of nitrogen from various sources and examine the influence of
N-source on the utilization of nitrogen by cotton plants.
Further, the
effect of other conditions, such as light and nitrogen deficiency, on
absorption and utilization were examined.
methods
Procedure for Growing Plants
Seeds of Gossypium hirsutum L. var. Acala 4-42 were placed in
8-oz
plastic cups two-thirds full of washed quartz sand.
The seeds
were then covered with another thin layer of sand and a thin layer of
vermiculite to prevent dessication and watered. The bottom of the cups
were perforated to allow easy drainage of excess water.
When seedlings reached the first true leaf stage of growth, the
plants were removed from the cups and the sand washed off the roots. The
plants were then placed in plastic-lined metal pots containing Hoagland's
(10) nitrogen-free nutrient solution, adjusted to pH 6.5 to 7.0.
Nitro­
gen was added as ammonium nitrate in the amount of 15 meq/1 of culture
solution.
The roots of the plants were suspended in the solution by wrapping
the lower portion of the stem just above the roots with cotton and placing
the plants in a slot cut in a piece of plywood.
The plywood cover was
made so it completely covered the container, thus preventing light from
reaching the culture solution. The culture solution was aerated by means
of a small tube from an air line extending into the solution.
Between
weekly changes of the solution, distilled water was used to make up for
transpiration losses.
The plants were allowed to grow for 30 days from
the time of emergence before they were used in the various studies.
15
16
Short-term Nitrogen Absorption Studies
The purpose of this experiment was to ascertain if a change in
the nitrogen level in the various nitrogen fractions of the plant could
be detected in a short absorption period.
A Percival growth chamber was
used and placed on a cycle which consisted of 12 hours of light at 35° C
and 12 hours of dark at 21° C.
Both fluorescent and incandescent light
was used, furnishing a light intensity of 1700 to 2000 foot-candles.
Plants were prepared as indicated and transferred to pots containing
nitrogen-free Hoagland's solution for 5 days. This period without
nitrogen was to produce a slight deficiency and to allow the adsorbed
nitrogen on the roots to be utilized as completely as possible, thus
minimizing the presence of any unwanted form of nitrogen in the experi­
ment.
Sets of three plants were transferred to each of eight 12-liter
pots containing nitrogen-free nutrient solution. Four of these pots re­
ceived 15 meq of nitrate-N as KNOg per liter of culture solution and the
other 4 pots received 15 meq of ammonium-N as (NH^^SO^ per liter of
culture solution.
To each of the pots 0.5 g of sulfanilamide was added
as a bacteriostatic agent to inhibit bacterial conversion of one form of
nitrogen to another.
At this time three plants were selected as a sample
to represent time zero (tQ) which was 3 hours into the light portion of
the cycle. Subsequent sets of plant samples were taken from each of the
nitrogen sources at 2, 6, 12, and 24 hours after tQ.
The tops were
separated from the roots 2 cm below the cotyledonary node, the roots
rinsed approximately one minute in distilled water, and each plant part
dried at 70° C. The experimental design was a split-split plot with
N-sources as whole plots, harvest times the first split, and plant part
the second split. There were two replications.
A second study was conducted to evaluate 0C-amino acids and urea
as sources of nitrogen for cotton plants.
same manner as before.
Plants were prepared in the
After 5 days without nitrogen, sets of three
plants were placed in pots containing 5 liters of culture solution. A
total of 17 meq of nitrogen from various 0(-amino acids was added to
each of two containers.
Another set of duplicate pots received a total
of 20 meq nitrogen each as urea.
one replication.
Three plants were harvested from each replication.
They represented t
cycle.
One pot from each N-source represented
which was 4 hours into the light portion of the
At the end of 24 hours the remaining plants were harvested.
Two meq N/1 from each of the following 0(-amino acids were used:
L-lysine, L-histidine, DL-threonine, L-valine, L-proline, L-serine,
L-leucine, L-iso-leucine, DL-alanine, L-phenylalanine, L-tyrosine,
L-tryptophan, L-methionine, L-'s-cystine, L-arginine, L-glutamine, and
L-asparagine.
This experiment was designed as a split-plot with N-source as
the whole plot and plant part the split. There were two replications.
During the experiment, samples of the culture solution were
taken at 6, 12, and 24 hours after tQ to evaluate the absorption of the
sources used. The culture solution was made up to the original volume
with distilled water and stirred; after 15 minutes a 50-ml aliquot was
18
taken from each pot at the specified harvest time.
The samples were
then frozen until they could be analyzed;
A third study involved prolonged light, prolonged dark, and the
12-hours light-12-hours dark cycle with ammonium-N, nitrate-N and am­
monium nitrate-N.
For that portion of the experiment conducted with the normal
light-dark cycle, four plants were placed in each pot which contained
4.5 liters of culture solution with the appropriate N-source added. The
plants had been without nitrogen for 5 days as usual.
There were two
pots for each N-source in each of three replications.
Just before the
plants were transferred, one plant was selected to represent tQ for each
of the N-sources. Two plants were harvested from each N-source at 8,
12, 20, and 24 hours after tQ. Time zero was 4 hours into the light
portion of the cycle.
off."
The 8-hour sample was taken just prior to "lights
The 12-hour sample was 4 hours into the dark period of the cycle,
and the 20-hour sample was just prior to "lights on."
The 24-hour sample
was 4 hours into the light period.
The plants used in the prolonged light and prolonged dark por­
tions of the experiment were prepared as previously described.
There
were four plants per pot and one pot for each source in each of three
replications.
regime.
One plant was harvested at 8 and 16 hours into each light
Two plants were harvested at 24 hours.
One plant representing
tQ was harvested at the beginning of the experiment.
The experimental design was a split plot with three replications.
Nitrogen sources were whole plots and times the split.
Samples of the culture solution were taken from replications I
and II in the light-dark regime for each of the harvest times except t .
Samples of the culture solution in the prolonged light and pro­
longed dark regimes were taken from three replications at the termina­
tion of the experiment.
The samples were taken and treated in the same
manner as described in the second experiment.
Deficiency Study
This study was conducted in the greenhouse to determine if the
response to ammonium, nitrate, and ammonium nitrate sources of nitrogen
was contingent upon the degree of nitrogen deficiency of the plants.
Three nitrogen regimes were used:
no deficiency, minus nitrogen 5 days,
and minus nitrogen 15 days. The plants used in this experiment were 35
days old at the beginning of the experiment.
Three plants were placed in each of three pots containing a
nitrogen-free nutrient solution.
These plants were to be grown in a
nitrogen-free culture solution for 15 days.
procedure was repeated.
days.
Five days later the same
These plants were to be nitrogen-free for 5
After 5 days, the latter plants and plants which had been main­
tained on a complete nutrient solution were transferred to pots contain­
ing the three different N-sources. They remained thus for a 24-hour
absorption period and were then harvested.
lution were taken.
Samples of the culuture so­
Plant and culture solution samples were handled as
previously described.
When the first group of plants reached 15 days
without nitrogen they were handled in the same manner as the other two
groups.
The reason for staggering the harvest date, and thus the age
20
of the plants, was to prevent a large disparity in the chronological age
of the plants in the experiment.
The experimental design was a split-plot with three replications.
Nitrogen regimes were the whole plot and nitrogen sources were the split.
Chemical Analyses
The oven dried plant tissue was ground with a small Wiley Mill
using a 40 mesh screen and collected in plastic vials.
A 0.0500 g
aliquot of the ground plant tissue was weighed out and placed in a 100-ml
Kjeldahl flask. The sample was then digested on a macro-Kjeldahl diges­
tion apparatus modified to accomodate semi-micro-Kjeldahl flasks as de­
scribed by Rauschkolb and Tucker (28).
The total Kjeldahl-N in the
sample was determined using Bremner's (3) modified semi-micro steam
distillation procedure.
Another portion of the ground sample was extracted with 80%
ethanol using the procedure outlined by Vonihof (36).
Aliquots of the extract were then used to determine the amount
of ammonium, nitrate and total reduced alcohol-soluble nitrogen present,
using Bremner's modified semi-micro steam distillation method.
The
frozen culture solution samples were allowed to thaw and then ammonium-N
and nitrate-N present was determined using steam distillation.
In addi­
tion, total nitrogen was determined on those samples taken from the
-amino acid and urea study.
RESULTS AND DISCUSSION
Preliminary Growth Chamber Studies
The purpose of this first study was to ascertain whether there
was a preference exhibited by the cotton plant for either an ammonium or
nitrate source of nitrogen.
At the same time the nitrogen level of
various fractions in the plant was followed to determine if changes in
the levels of these fractions could be detected in a short period of
time.
Both the tops and the roots were studied to observe the relative
change of nitrogen level in these plant parts with respect to the dif­
ferent N-sources.
With ammonium as the N-source the total Kjeldahl-N in the tissue
decreased significantly in the roots (fig. 1).
After an initial de­
crease, the level increased sharply and then gradually increased to the
level prior to exposure to the ammonium source.
With nitrate as an
N-source the total Kjeldahl-N in the roots continued to increase during
the 24 hours of the experiment but never reached a level which was sig­
nificantly higher than the initial level.
In the tops (fig. 1) the total Kjeldahl-N remained essentially
unchanged for the duration of the experiment regardless of N-source.
The nitrogen level of this fraction in the tops with the ammonium Nsource did exhibit the same decrease as the roots, although the decrease
could not be statistically detected.
The total alcohol-soluble reduced-N fraction (fig. 1) in the
roots increased significantly in 12 hours, essentially during the light,
\
\
22
LIGHTS
ON
LIGHTS
OFF
30
-o
-1
.o—
LU
S
cn
25
lh-
z:
<
_i
q.
>-
Cd
q
E
cn
E
E
cn
TOP
z
UJ
CD
o
cn
i-
z
Sjj = 2.06 mgm
0
8
16
TIME—HOURS
FIG. I TOTAL KJELDAHL-N IN PLANT TISSUE
TOPS AND ROOTS
24
23
SOURCE
TOPS:
SOURCE
NH 4 -N &
a
NO, -N a
A
o
2 •
ROOTS:
NH^-N o—o
NOj-N
o
AMMONIUM -N
>|
Sx = 0.359 mgm
NITRATE -N
Sx = 1.054 mgm
10
TOTAL ALCOHOL
j
SOLUBLE REDUCED-N
'
S5 = 0.612 mgm
LIGHT^ ON
LIGHTS OFF
l_l
8
16
TIME—HOURS
FIG. 2
AMMONIUM,NITRATE, AND TOTAL ALCOHOLSOLUBLE REDUCED NITROGEN FRACTIONS —
TOPS AND ROOTS
o
24
with nitrate as the N-source.
No further increase occurred during the
remaining 12 hours of the experiment which was mainly in the dark.
Ap­
proximately one-half of the increase which occurred in the total
Kjeldahl-N in the root tissue of plants grown with nitrate-N was the
result of an increase in the total soluble reduced-N in the roots from
the same plants.
In the tops of plants grown with nitrate-N there was a slight
decrease in the total soluble reduced-N, but then the nitrogen level of
this fraction increased until there had been a significant increase
over the 6 hour harvest time. The nitrogen level was not significantly
higher at the termination of the experiment than it was at tQ.
In the ammonium fraction (fig. 2) a significant increase occurred
in the roots with nitrate as the N-source at the 6-hour harvest time.
Inspection of the data showed a large disparity between replication I
and II for the 6-hour harvest time value.
Therefore, it is possible the
mean value for the harvest time was unrealistically high and that no
changes occurred in the ammonium fraction.
In the nitrate fraction (fig. 2) an increase occurred in the
roots of plants grown with nitrate as the N-source.
A significant in­
crease had occurred in the first 12 hours of the experiment and most of
that increase was in the first two hours.
The nitrogen level increased
for essentially the whole 24 hours of the experiment.
In the tops of plants the nitrate fraction remained constant
regardless of N-source.
Also, in the roots of plants grown with ammon-
ium-N the nitrate level remained unchanged.
25
The slight decrease in the total soluble reduced-N in the tops
with nitrate as the N-source possibly was due to a lag in the reduction
of nitrate to ammonium in the plant tissue.
The translocation of the
soluble reduced-N to the roots and its conversion to protein could have
proceeded at a faster rate than the soluble reduced-N fraction in the
tops was replenished.
As the protein deficit in the roots was relieved
then the total soluble reduced-N in the tops increased.
Holley et al. (11) found that the formation of organic nitrogen
from nitrate in the plant proceeded at a slower rate than the conversion
of ammonium to organic nitrogen.
Syrett(30) found that respiration of
polysaccharide and the nitrate reduction were competing for the NADH
present in the plant. These studies are corroborative and tend to sup­
port the previous statements.
The increase in the nitrate level of the roots of plants grown
with nitrate-N may be explained on the basis of experiments conducted by
Lycklama (19). He concluded that nitrate reduction and accumulation were
two different processes.
Therefore it would be possible for reduction
and accumulation to be occurring simultaneously.
Since a decrease was observed in the total Kjeldahl-N of the
tops and roots of plants grown with ammonium-N and not in the total
soluble reduced-N the decrease must have occurred in the alcohol insoluble
fraction of the plant tissue.
As indicated in the literature review the
alcohol insoluble fraction has been construed by several investigators as
a measure of the protein content of plant tissue.
26
If protein is degraded without a concomitant increase in other
nitrogen pools in the plant then an actual loss of nitrogen must have
occurred.
One way nitrogen could have been lost was exudation into the
culture solution.
Although the amount of nitrogen lost from the plants
represented a considerable amount from the plant's standpoint, this
quantity could not be measured accurately when diluted by large volumes
of culture solution.
The subsequent increase was the result of either re-utilization
of exuded nitrogen, absorption and utilization of ammonium, or a com­
bination of these.
It is evident that with nitrate as the N-source the nitrogen
level of all fractions in each plant part was relatively higher than
with ammonium as the N-source.
One exception to this was the ammonium
fraction in the roots with ammonium as the N-source.
Also, the nitrogen
level in the roots for each of the fractions was higher than in the tops.
Another study was conducted in the growth chamber to evaluate
-amino acids and urea as nitrogen sources for cotton plants.
The
only nitrogen fraction in which a significant change was observed was
the nitrate fraction (Table 1). With 0C-amino acids as the N-source the
nitrate level in the tops decreased below the value obtained for the
control.
The value for the total Kjeldahl-N content of the tops of
plants grown with urea as the N-source appeared lower than other values
(Table 1); however, the difference could not be detected statistically.
In Tables 2 and 3 data are presented showing the amounts of
nitrogen absorbed from the different N-sources and the amounts of
27
TABLE 1
THE AMOUNT OF NITROGEN IN VARIOUS FRACTIONS OF TOPS
AND ROOTS AS INFLUENCED BY ORGANIC N-SOURCES
Nitrogen
Source
Ammonium
Nitrogen Fraction**
Total Soluble
Nitrate
Reduced-N
Total
Kjeldahl-N
*
Control
^C-amino
acids
Urea
sx
T
0.17 a
1.58 c
3.81 a
23.85 a
R
0.17 a
0.58 ab
4.24 a
24.56 a
T
0.23 a
0.26 ab
3.50 a
22.85 a
R
0.08 a
0.20 a
4.83 a
23.29 a
T
0.28 a
1.11 be
3.68 a
19.10 a
R
0.14 a
0.53 ab
4.07 a
24.64 a
0.0525
0.194
0.820
2.08
*T = Tops, R = Roots.
**Values followed by the same letter within a given fraction are not
significantly different.
28
TABLE 2
. CONCENTRATION OF AMMONIUM AND NITRATE IN THE CULTURE
SOLUTION CONTAINING ORGANIC N-SOURCES
Sampling
Time
Hours
6
Nitrogen Sources
oC-Amino Acids
Urea
Ammonium
Nitrate
Ammonium
Nitrate
mgm N/liter
3.90 c*
0.81 ab
0.44 ab
0.88 ab
12
7.40 d
0.32 ab
0.20 ab
0.68 ab
24
8.60 e
0.97 b
0.08 a
0.36 ab
*Values are means of 2 replications. In the table values followed by the
same letter are not significantly different; s- = 0.246 mgm.
TABLE 3
NITROGEN ABSORBED FROM ORGANIC N-SOURCES
Sampling
Time
Hours
6
Nitrogen Sources
Amino Acids
mgm N absorbed/pot
Urea
75*
44
12
65
42
24
48
34
*Values are means of 2 replications.
Values are not significantly different.
ammonium-N and nitrate-N present in the culture solution, respectively.
When the amino acids used in this experiment were placed in the culture
solution there was a build-up of ammonium in the solution.
This build­
up was due to the hydrolysis of the amide forms of the amino acids. The
amount of nitrogen absorbed from the solution was approximately twice
that which could be accounted for on the basis of complete absorption
of the hydrolyzed nitrogen from the amides.
One could infer from these
data that plants can absorb amino acids and urea as intact molecules.
Ghosh and Burris (7) on the basis of their studies implied that
amino acids were assimilated by plants as intact molecules. The same
view is supported by Virtanen and Linkola (34).
Effect of Light Regime on Absorption
The absorption studies conducted with ammonium and nitrate as
N-sources showed that changes could be detected in the nitrogen level of
various fractions during short term absorption periods.
Therefore,
studies were designed to evaluate the influence of light on the absorp­
tion of the inorganic N-sources (ammonium, nitrate and ammonium nitrate)
Three different experiments were designed to study the interac­
tion between N-source and light.
These experiments differed from each
other only in the length of time the plants were grown in the light or
dark period.
The only significant differences in absorption with respect to
N-source (Table 4) occurred in the prolonged dark period.
The disappear
ance of nitrogen from the culture solution was greatest with ammonium as
the N-source and least with ammonium nitrate as the N-source.
The same
TABLE 4
ABSORPTION OF NITROGEN FROM DIFFERENT N-SOURCES IN
NUTRIENT SOLUTION AS INFLUENCED BY LIGHT REGIME
Sources
Nitrate
Ammonium
LightDark*
.
8
77
91
112
12
97
129
142
20
145
135
142
24
141
Light*
172
Dark^
216 c
LightDark
Light
Dark
mgm N absorbed per pot
Ammonium Nitrate
LightDark
Light
Dark
Sampling
Time
Hours
135
130
143 b
130
109
103 a
*Light-dark values: means of 2 replications, sources are not significantly different.
+Light values: means of 3 replications/ no significant differences.
#Dark values: means of 3 replications, differences are significant. Values followed by the same
letter are not significantly different; s- = 3.88 mgm.
31
trend was observed in the prolonged light regime; however, significant
differences were not detected due to sample variability.
In the light-dark regime no significant difference was detected
with respect to N-source.
Also, a significant source by time interaction
was not detected, implying the absorption rates were the same for the
N-sources.
A significant difference was found in the absorption rates
without regard for N-source.
The means for sampling time as separated
by the Duncan multiple range test are shown as follows:
8-hr
12-hr
20-hr
24-hr
93 mgm
123 mgm
141 mgm
135 mgm
a
b
Sjj = 8.63 mgm
Light regime did not have any effect on the absorption of nitrate
by plants from the culture solution. On the other hand, absorption of
ammonium was enhanced by either prolonged light or dark, more so in pro­
longed dark.
The absorption of ammonium nitrate appears to be adversely
affected by prolonging the light or dark period.
Effect of Light Regime on Utilization
The data obtained from the analyses of the plant parts for vari­
ous nitrogen fractions was subjected to statistical analyses. The statis­
tical procedure used was a split-split plot technique. The fractions
were analyzed separately with the N-sources as whole plots.
The first
split was harvest times and the plant parts constituted the second split.
32
The means of the various factors and their interactions were separated
using Duncan's multiple range test.
According to Steel and Torrie (29)
the separation of means by this method may be used regardless of the in­
significance of the F ratio.
Light-Dark Cycle
Changes in the various fractions as influenced by N-source and
harvest time for both tops and roots may be seen in figures 3 through 7.
In the ammonium fraction a significant difference was found
between harvest times without regard to plant part or N-source.
In the
first 8 hours of the experiment an increase in the nitrogen level of
this fraction occurred, after which the level did not change.
The nitrogen level in the roots was significantly higher than in
the tops.
In addition there was a significant plant part by N-source
interaction, implying tops and roots responded differently to the N-source.
With nitrate as the N-source the ammonium level in the tops in­
creased steadily for the first 20 hours of the experiment; the level at­
tained in this time was significantly higher than the initial level.
With ammonium and ammonium nitrate N-sources no significant changes were
detected.
In the roots with ammonium nitrate as the N-source a significant
increase occurred in the first 8 hours of the experiment and then for
the remainder of the time the level steadily decreased.
With ammonium
as the N-source the ammonium level in the roots gradually increased
until a significant increase was observed at the 20-hour harvest time.
The*ammonium^ fraction remained constant for the whole experiment with
33
TOPS
.8
.7
.6
.5
.4
.3
.2
i
.1
Sx = 0.0837 mgm
0
8
0
24
16
TIME-HOURS
LIGHT-DARK CYCLE
nh4
A
A
no^
o
o
NH4NO3 *
*
ROOTS
o
S x = 0.0837 mgm
2
LIGHTS OFF
LIGHTS ON
24
TIME—HOURS
FIG.3
AMMONIUM FRACTION OF LIGHT-DARK CYCLE PLANTSTOPS AND ROOTS
34
TOPS
NH,
NO.
o
-o
NH. NO, X
-X
Sx = 0.396 mgm
0
8
24
16
TIME-HOURS
ROOTS
SX
= 0.396 mgm
o—
LIGHTS OFF
0
8
,
16
LIGHTS. ON
24
TIME-HOURS
FIG. 4
NITRATE FRACTION FOR LIGHT-DARK CYCLE PLANTSTOPS AND ROOTS
35
TOPS
NH4
NO3
NH4NO3
A
A
o
-o
X
x
^o>
0.926 mgm
8
16
TIME-HOURS
ROOTS
Sx
=
0.926 mgm
LIGHTS ON
LIGHTS OFF
TIME
FIG. 5
HOURS
TOTAL ALCOHOL-SOLUBLE REDUCED - N FOR
LIGHT-DARK CYCLE PLANTS - TOPS AND ROOTS
36
NH,
NO
34
o
o
30
ZD
cn
25
cl
20
- or
LINE OMITTING LOW
VALUES IN REP. 3H
o>
cf»
.53 mgm
LIGHTS OFF
0
8
LIGHTS ON
16
24
TIME—HOURS
FIG. 6
TOTAL KJELDAHL-N IN DRY PLANT TISSUE FROM
LIGHT-DARK CYCLE PLANTS - TOPS
nh
no
o—o
nh/, no-,
x-—*
sx = 1.53 mgm
lights off
0
8
lights on
16
24
TIME-HOURS
FIG. 7
TOTAL KJELDAHL-N IN DRY PLANT TISSUE FROM
LIGHT-DARK CYCLE PLANTS - ROOTS
the nitrate-N source.
A possible reason for the increases observed in
this fraction for both the ammonium and ammonium nitrate N-sources in
the roots could be the adsorption of ammonium onto the surface of the
roots.
In the nitrate fraction significant differences were detected
in harvest times, plant part, and plant part by N-source interaction.
An initial increase was observed at the 8-hour hcirvest time after which
no other changes were detected.
The nitrate level in the tops was higher than in the roots.
Nitrogen source had little effect on the nitrate fraction in the tops
as compared with the roots.
Root nitrate was increased by the nitrate
and ammonium nitrate sources.
The higher nitrate level in the roots of plants supplied N-sources
consisting wholly, or in part, of nitrate, could be the result of infusion
with nitrate into the apparent free space of the roots.
During the first 12 hours of the experiment, mainly in the light,
an increase was detected in the nitrate level of roots of plants grown
with ammonium nitrate.
In the next 8 hours, during the dark, a decrease
occurred, and then in the remaining 4 hours, in the light, another in­
crease was observed.
With nitrate as the N-source the nitrate level in
the roots steadily increased for the first 20 hours of the experiment.
In the last 4 hours, in the light, the nitrate level appeared to decrease,
although not significantly.
With ammonium as the N-source, the nitrate
level did not change in the roots.
In the tops, the nitrate level with ammonium nitrate as the Nsource did not change.
With nitrate-N a significant increase occurred
in the first 8 hours of the experiment; a slight decline occurred in the
dark, thereafter the level remained essentially unchanged.
With ammonium
as the N-source the nitrate level of the tops had increased significantly
at the termination of the experiment.
This observation cannot be ex­
plained on the basis of data obtained from these experiments.
The result
was unexpected since oxidation of ammonium-N to nitrate-N is contrary to
generally accepted metabolic pathways in higher plants.
The nitrogen level in the total soluble reduced-N fraction ap­
peared to increase with time} however, no significant changes were de­
tected.
The nitrogen level in this fraction in the tops of the plants
grown with ammonium-N showed a decrease in the first 4 hours of the dark
period and then an increase for the remaining time of the experiment.
The roots of these same plants showed a steady increase in the'total" "•
soluble reduced-N during the dark portion of the cycle.
In the tops the
total soluble reduced-N of plants grown with nitrate-N appeared to attain
and maintain a higher level than with the other N-sources.
The total Kjeldahl-N fraction in the tops of plants grown with
nitrate and ammonium-N sources had significantly increased in 20 hours
and did not change from that level during the last 4 hours of the experi­
ment.
Most of the increase was occurring in the alcohol insoluble frac­
tion (protein) since there was no increase in the total soluble reduced-N
fraction.
The major portion of the increase which occurred with the
ammonium N-source was during the last 8 hours of the dark portion of the
cycle.
In the tops of plants grown with the nitrate-N source approxi­
mately one-half the increase occurred during the initial 8 hours of light
and the other half during the dark portion of the cycle.
40
No change occurred in this fraction in the tops of plants grown
with ammonium nitrate as the N-source.
An alternative line was plotted for the ammonium nitrate source.
In the third replication the values for t^ and t2 were considerably lower
than in the other two replications.
The roots of plants grown with ammonium and ammonium nitrate
N-sources exhibited similar changes with respect to time.
However, dur­
ing the dark period of the cycle a significant increase occurred in the
total Kjeldahl-N content of the roots of plants grown with ammonium
nitrate-N whereas with ammonium as the N-source the total Kjeldahl-N
content of the tissue returned to the level prior to exposure to
ammonium-N and remained unchanged thereafter.
The increase in the protein concentration in the tops of the
plants grown with ammonium and ammonium nitrate N-sources may,in part or
totally, account for the decrease in the same fractions in the roots,
via translocation of metabolites.
However, the loss of nitrogen into the
culture solution as discussed previously could have accounted for part of
the loss.
An increase in the total Kjeldahl-N in the roots with nitrate as
the N-source occurred in the first 8 hours of the experiment, which was
in the light.
Approximately one-half of the protein which was gained
was lost in the first 4 hours of the dark period, and then the level
remained essentially unchanged thereafter.
Since the loss in the roots
was concomitant with a gain in the protein level of the tops the nitrogen
must have moved to the tops via a translocatable metabolite. It was not
41
likely in this case that any large amount of nitrogen was being exuded
into the culture solution.
Aerial portions of the cotton plant were used exclusively in
studies conducted by Phillis and Mason (26, 27) and Maskell and Mason
(20, 21).
They found the nitrogen content of the plant parts used was
fluctuating with diurnal changes.
They inferred from these data that
translocation from the leaves to the roots was occurring during the
night and that translocation from the roots to the tops was occurring
during the day.
It appears, from the data obtained in this experiment, that the
relative changes in various nitrogen fractions of the tops and roots are
influenced by N-source as well as diurnal changes.
Prolonged Light
The ammonium fraction (fig. 8) in the tops did not change with
respect to time or N-source.
In the roots no significant changes were
detected with either ammonium or nitrate N-sources.
However, with
ammonium nitrate as the N-source an increase occurred in the first 8
hours after which the nitrogen level remained essentially constant.
Be­
cause of the low t value obtained for the ammonium nitrate N-source a
....
o
significant increase was observed at the termination of the experiment;
however, on the whole, a significant difference was not detected among
the ammonium levels obtained for the various N-sources.
In this experi­
ment as in past studies the overall mean value obtained for the roots
was greater than that obtained for the tops.
42
TOPS
.8
.7
.6
.5
lj
ZD
CD
CD
\—
17
<
.4
.3
.2
-o-
Sx = 0.0619 mgm
.1
0
8
_J
q_
>ck
o
E
o>
16
TIME
24
HOURS
LIGHT CYCLE
4——A
NH4
o
o
NO3
NH4NO3
X
X
ROOTS
• o— -
S£ = 0.0619 mgm
i
»
8
16
24
TIME-HOURS
FIG. 8
AMMONIUM FRACTION OF LIGHT CYCLE PLANTS TOPS AND ROOTS
43
The same general change in the tops with respect to time was ob­
served in the nitrate fraction (fig. 9) for each of the N-sources.
How­
ever, with the nitrate N-source the increase was significant during the
first 8 hours of the experiment, whereas with the other N-sources signi­
ficant changes were not detected.
A significant decrease occurred dur­
ing the next 8 hours with nitrate as the N-source while no significant
change was observed for the ammonium and ammonium nitrate sources.
The nitrate level was significantly higher at the termination of
the experiment than it was initially with the ammonium nitrate and nitrate
N-sources.
In the roots, no significant changes with respect to time or
N-source were found. The nitrate level in the roots was significantly
lower than in the tops.
The nitrate fraction in the tops and roots was influenced in the
same manner by both the nitrate and the ammonium N-sources, although the
magnitude of the changes was considerably different.
With ammonium
nitrate as the N-source, the relative changes in the tops and roots were
dissimilar.
The total soluble reduced-N fraction (fig. 10) did not exhibit
any significant changes with respect to time in the tops or roots when
ammonium was the N-source. When nitrate was the N-source a significant
increase occurred in the roots at the 16 hour harvest time; however, no
significant change was detected in the tops.
With ammonium nitrate as
the N-source the nitrogen level of the total soluble reduced-N fraction
increased in both the tops and the roots during the first 8 hours of
44
TOPS
4
Ui
z>
co
cd
//
s
_•
x
<
_l
cl
Sx = 0.396 mgm
>
c£
o
i
0
8
S
i
.i
24
TIME-HOURS
E
d>
16
4
E
cr>
E
ROOTS
NH4
NO3
z:
uj
cd
o
NH 4N0J
o
o
x
x
en
j—
2
sx
8
= 0.396 mgm
16
TIME —HOURS
FIG. 9
NITRATE FRACTION OF LIGHT CYCLE PLANTS —
TOPS AND ROOTS
45
6
NH 4
A
A
N0 3
O
o
NH 4 N0 3
X
x
TOPS
5
4
3
-o
Sx = 0.586 mgm
0
8
0
16
24
TIME-HOURS
o
s
ROOTS
"Z.
UJ
CD
o
•c£
h
"o
O'
Sx = 0.586 mgm
0
8
16
24
TIME-HOURS
FIG. 10
TOTAL ALCOHOL-SOLUBLE REDUCED-N OF LIGHT
CYCLE PLANTS — TOPS AND ROOTS
46
the experiment.
The level of this fraction in the tops did not change
significantly thereafter.
In the roots a significant decrease occurred during the last 8
hours of the experiment; however, the nitrogen content of the total
soluble reduced-N fraction was higher than the t
value.
A general increase appeared to occur in the total Kjeldahl-N
fraction (fig. 11) in the tops during the first 8 hours of the experi­
ment regardless of N-source.
After the initial increase the total
Kjeldahl-N in the tops with ammonium nitrate and nitrate N-sources re­
mained essentially unchanged for the remainder of the experiment. With
ammonium as the N-source the total Kjeldahl-N decreased significantly
from a maximum level at 8 hours and remained unchanged for the last 8
hours of the experiment.
With nitrate as the N-source the total Kjeldahl-N appeared to
change in the roots (fig. 12) in the same manner as observed in the tops.
The total Kjeldahl-N in the roots exhibited a significant decrease in the
first 8 hours of the experiment when ammonium and ammonium nitrate were
the N-sources. The total Kjeldahl-N concentration in the roots continued
to decrease in the next 8 hours with ammonium nitrate-N, thereafter in­
creasing in the final 8 hours of the experiment to a level equal to tQ.
With ammonium as the N-source, total Kjeldahl-N increased steadily for
the last 16 hours until it had returned to the level present at the ini­
tiation of the experiment.
As indicated in previous experiments, when changes occurred in
the total Kjeldahl-N fraction all or part of the change appeared to
47
a
NH
44
—o
NO.
x
40
LLI
13
CO
c
r-
a
x
36
32
i-
z:
<
q_
>-
0q
24
£
o
16
o
Sv
43 mgm
4 •
0
,
0
'
8
16
1
"
24
TIME—HOURS
FIG. II- TOTAL KJELDAHL-N IN DRY PLANT TISSUE OF
LIGHT CYCLE PLANTS — TOPS
48
44
40
36
32
~- o —
a
24
NH4
NO3
NH, N0_
o—o
x
x
sx
0
8
s
1.43 mgm
16
24
TIME-HOURS
FIG. 12 TOTAL KJELDAHL-N IN DRY PLANT TISSUE OF
LIGHT CYCLE PLANTS - ROOTS
49
occur in the protein concentration of the tissue.
This also seems to be
the case in this study.
With ammonium as the N-source there appeared to be an overall
loss of nitrogen from the plants since changes in the total Kjeldahl-N
of the roots and tops were not commensurate.
Also, since ammonium was
absorbed from the solution, which implies utilization by the plant, the
plant could have been concurrently assimilating ammonium and exuding
other nitrogenous compounds into the culture solution.
When the N-source was ammonium nitrate, protein in the roots
appeared to be degraded into a more mobile form of nitrogen which was
then translocated to the tops.
As the plants reached an equilibrium
between absorption, translocation, and utilization the protein level in
the roots increased and the soluble reduced-N pool decreased.
Increases occurred in the total Kjeldahl-N and nitrate fractions
without similar increases in the total soluble reduced-N when nitrate
was the N-source.
This implies that protein is being formed rapidly by
the plant and nitrate was concomitantly accumulating in the plant. This
effect has been observed previously and lends support to the conclusion
of Lycklama (19).
Prolonged Dark
A significant change was detected in the ammonium fraction (fig.
13) with respect to harvest time.
At the 16-hour harvest time, the ni­
trogen level in the ammonium fraction was higher than at any other time.
This was an overall effect without regard to the N-source or plant part.
Also, nitrogen content in the ammonium fraction in the roots was
50
lu
z>
cn
cn
Sx = 0.0597 mgm
h-
8
z:
<
16
24
TIME-HOURS
_i
q_
DARK
CYCLE
>
cn
q
E
cn
s
nh4.
—a
NO 3
o—o
NH 4 N0 3
x
X
£
cn
e
1
2
lli
cd
o
or
i—
ROOTS
Sv = 0.0597 mgm
*
8
16
24
TIME—HOURS
FIG. 13
AMMONIUM FRACTION OF DARK CYCLE PLANTSTOPS AND ROOTS
51
significantly higher than in the tops.
As pointed out previously this
could be the result of adsorbed Nh£ on the surface of the roots of plants
grown with N-sources consisting, totally or in part, of ammonium-N.
Significant increases in the ammonium fraction of the roots were
detected when ammonium and ammonium nitrate were the N-sources.
The
nitrogen level of this fraction in the roots essentially did not change
with the nitrate-N source.
The tops of plants grown with the ammonium nitrate N-source ex­
hibited a significant increase in the ammonium fraction at the 16-hour
harvest time; however, a decrease of the same magnitude occurred in the
final 8 hours of the experiment.
Essentially no change occurred in this
fraction when plants were supplied nitrogen from the ammonium or nitrate
N-source.
The main effects, harvest time and plant-parts, were found to
contain significant differences with respect to the nitrate fraction
(fig. 14).
The overall effect of added nitrogen was to increase the
nitrate level more in the tops than in the roots.
A significant increase
with respect to time occurred during the first 8 hours of the experiment,
after which the nitrate level remained constant.
In general, the nitrate level of the tops followed the same
pattern with time. With the ammonium nitrate N-source, no significant
changes occurred in the nitrate fraction of the tops.
At the 8-hour and 24-hour harvest times significant increases
over the t
nitrate level were found in the nitrate fraction when am­
monium and nitrate were the N-sources.
52
4 f-
NH,
TOPS
NO.
o_
NH 4 N0 3
k-'-X
o
u
3
(f)
tn
h
2:
<
_i
CL
Sx = 0.342 mgm
>-
cc
q
0
'
0
E
8
1
24
16
TIME—HOURS
o
e
cj>
e
\
1
4
"
ROOTS
iLi
CD
o
Cd
i—
2:
1
—o
—A
Sx= 0.342 mgm
8
16
24
TIME—HOURS
FIG. 14
NITRATE FRACTION OF DARK CYCLE PLANTSTOPS AND ROOTS
53
In the roots the nitrogen level of the nitrate fraction was sig­
nificantly higher at the 16-hour and 24-hour harvest times than at tQ
when nitrate was the N-source.
No significant changes were detected when
ammonium and ammonium nitrate were the N-sources.
The increase in the nitrate fraction of the tops when ammonium
was the N-source cannot be explained from data available in this experi­
ment.
As pointed out previously the oxidation of ammonium-N to nitrate-N
is contrary to what is generally known about nitrogen metabolism in higher
plants.
Analyses of aliquots of the culture solution indicated that when
(NHj)2SOg was used as the N-source nitrate-N concentration of the culture
solution was less than 1 mg/1.
Therefore, it was unlikely that nitrate
was present at a concentration level which could effect a change of the
magnitude observed.
Also, translocation of root nitrate to the tops
would not account for the amount involved.
The total soluble reduced-N fraction (fig. 15) was significantly
higher at the 16-hour harvest time than at tQ in both the tops and the
roots when ammonium nitrate was the N-source.
However, during the last
8 hours of the experiment the total soluble reduced-N concentration de­
creased to a value not significantly different from the t
value.
No significant changes occurred in the total soluble reduced-N
fraction of either the tops or roots when ammonium or nitrate were the
N-sources.
The only change in the total Kjeldahl-N fraction (fig. 16) in
the tops which could be detected statistically with respect to tQ was
the increase which occurred at the 16-hour harvest time with nitrate as
54
TOPS
NH4NO3
»x = 1.65 mgm
8
16
TIME-HOURS
ROOTS
Sw = 1.65 mgm
8
16
TIME—HOURS
FIG. 15
TOTAL ALCOHOL-SOLUBLE REDUCED-N OF DARK
CYCLE PLANTS — TOPS AND ROOTS
55
35
32
L'J
28
24
nh.
o
NO
o
lj
Sj{ = 2.84 mgm
0
8
16
24
TIME—HOURS
FIG. 16
TOTAL KJELDAHL-N IN DRY TISSUE OF DARK
CYCLE PLANTS - TOPS
the N-source.
A significant change was detected in the total Kjeldahl-N
fraction in the roots (fig. 17) between the 8-hour and 16-hour harvest
time with ammonium as the N-source.
Also, without regard to other main
effects the total Kjeldahl-N concentration in the root tissue was signi­
ficantly higher than in the tops.
An exchange of nitrogen appeared to be occurring between the tops
and roots of plants grown with ammonium as the N-source.
Also, ammonium
was being absorbed from the culture solution and apparently utilized.
Since ammonium-N was being absorbed from the culture solution without a
subsequent increase in the nitrogen content of the plant tissue the im­
plication is that nitrogen was being lost from the plant in the form of
some soluble reduced-N compound. This in turn implies that assimilation
of ammonium-N and loss of soluble reduced-N compounds to the culture
solution were occurring simultaneously.
With ammonium nitrate as the N-source significant changes were
not detected in the total Kjeldahl-N; however, the total soluble
reduced-N, which constitutes a portion of the total Kjeldahl-N, did in­
crease in both the tops and roots. The increase in the total soluble
reduced-N fraction may be accounted for in two ways:
(1) protein in the
tops and roots was degraded into an alcohol soluble form of nitrogen,
and (2) as ammonium nitrate was assimilated, concomitant loss of soluble
reduced-N from the plants was occurring.
Some rearrangement of the nitrogen occurred between the tops and
roots of plants grown with nitrate as the N-source; however, changes were
not of the same magnitude as with the other N-sources.
It appeared that
57
35
32
289
24
20
NH.
l'j
NO
<S>
o
cc
h
'X = 2.84 mgm
8
0
16
24
TIME—HOURS
FIG.
17
TOTAL KJELDAHL-N IN DRY TISSUE OF DARK
CYCLE PLANTS — ROOTS
58
during the last 8 hours of the experiment the decrease in the protein
content of the tops was greater than the increase in the roots.
This,
again, could result in the exudation of soluble reduced-N into the cul­
ture solution.
Effect of Nitrogen Deficiency on Absorption and Utilization
Absorption
Samples of the culture solution from the experiment conducted in
the greenhouse were analyzed for ammonium and nitrate.
The unreplicated
data showing the disappearance_of nitrogen from the culture media was
given in Table 5.
Plants which had been nitrogen-free for 15 days (minus-N 15 days)
absorbed a much greater amount of nitrate-N from the culture solution than
ammonium-N or ammonium nitrate-N.
There was no difference between the
total amount of nitrogen absorbed from the two latter N-sources.
The non-deficient plants (complete) and the plants deficient for
5 days (minus-N 5 days) appeared to absorb ammonium-N and nitrate-N
equally well.
More total nitrogen was absorbed when the source was am­
monium nitrate from which a greater amount of nitrate-N was absorbed.
Utilization
Perhaps the most important effect in the ammonium fraction (fig.
18) was a change which occurred with "Respect to nitrogen regime. The
ammonium fraction in the non-deficient plants was significantly higher
than in plants which had been grown without nitrogen.
There was no dif­
ference between the ammonium levels of the control plants which had been
grown without nitrogen for 5 days and 15 days.
59
TABLE 5
THE EFFECT OF NITROGEN SOURCE AND DEFICIENCY ON ABSORPTION
Milligrams of Nitrogen
absorbed per pot as;*
Nitrogen
Regime
Nitrogen
Source
Complete
nh4
Ammonium
205
n03
Minus N
5 days
nh4n03
110
nh4
180
N0_
3
Minus N 15 days
nh.n0_
4 3
100
nh4
235
n03
nh4n03
*Values are unreplicated.
Nitrate
100
Total
205
205
205
150
260
180
220
220
155
255
235
365
365
130
230
60
ezzza = CONTROL
K2S - NH4
t=l = NO 3
\EE
=NH4N03
TOPS
1.5
1.0
Q5
CL
cr
0
ROOTS
CO
lil
o
COMPLETE
MINUS N
5 DAYS
MINUS N
15 DAYS
fig. 18 ammonium fraction for different nitrogen
regimes and sources-tops and roots
G1
In the non-deficient plants the ammonium fraction was signifi­
cantly lower in the tops when nitrate was the N-source, than with any of
the other N-sources.
The ammonium fraction in the roots of plants grown
with ammonium as the N-source was significantly higher in the roots of
the control plants.
The ammonium level in the roots of plants grown
with ammonium nitrate and nitrate-N sources was not different from the
ammonium level in the roots of control plants.
When plants had been grown in a nitrogen-free culture solution
for 5 days prior to placing them in a culture solution containing the
various N-sources no significant change was observed in the ammonium
fraction in the tops. The ammonium fraction in the roots of plants
grown with the ammonium N-source was higher than the ammonium fraction
in the roots of the control plants.
In the plants with the minus nitrogen pretreatment for 15 days
the amonium fraction in the roots was not affected significantly by
various N-sources.
The ammonium fraction in the tops of plants grown
with a nitrate N-source was significantly higher than under conditions
of ammonium or ammonium nitrate-N sources.
The main effects, nitrogen regime,N-source, and plant part, were
all highly significant in the nitrate fraction (fig. 19).
level in the tops was greater than in the roots.
The nitrate
The nitrate fraction
in plants with adequate nitrogen was higher than in plants which had been
nitrogen deficient.
When nitrate was the N-source the nitrate fraction
in the plants was higher than the control plants or plants supplied with
ammonium and ammonium nitrate N-sources.
62
P77771
= control
= nh 4
en
=no 3
m
=nh 4 no 3
i5{-
tops
15
roots
10
J
complete
fig. 19
minus n
5 days
minus n
15 days
nitrate fraction for different nitrogen
regimes and n-sources -tops and roots
63
In the tops of the non-deficient plants no significant differ­
ences could be detected, although the nitrate fraction appeared to be
lower when ammonium was the N-source. The nitrate fraction of the roots
was higher in the plants grown with nitrate-N than in the control plants
and in plants grown with ammonium-N.
No significant changes occurred in the nitrate fraction of plants
which had been grown 5 days without nitrogen.
The nitrate fraction increased significantly in the tops of
plants grown 15 days without nitrogen and then supplied a nitrate
N-source. The nitrate fraction in this instance was significantly higher
in the control plants or plants supplied with ammonium or ammonium nitrate
N-sources.
Also, the nitrate fraction was significantly higher in the
roots of plants grown with a nitrate N-source than in the roots of plants
grown with an ammonium N-source, but not significantly higher than in the
control plants or plants grown with an ammonium nitrate N-source.
Differences with respect to nitrogen regime were higher signifi­
cantly in the total soluble reduced-N fraction (fig. 20).
Also, a highly
significant nitrogen regime by N-source interaction was found which im­
plies that the total soluble reduced-N fraction was influenced differ­
ently by N-sources with the various nitrogen pretreatment regimes. The
total soluble reduced-N fraction was higher in the non-deficient plants
than in the nitrogen deficient plants.
Also, the total soluble reduced-N
fraction was higher in plants grown 5 days without nitrogen than in
plants grown 15 days without nitrogen; however, the difference in the
total soluble reduced-N fraction was not as great between the two nitrogen
64
EZZZ3
5
bsa
= nh4
CONTROL
CD = N03
TZZ3 =NH4N03
TOPS
1
ROOTS
COMPLETE
MINUS N
5 DAYS
MINUS N
15 DAYS
fig. 20 total alcohol-soluble reduced-n fraction
for different nitrogen regimes and n-sourcestops and roots
65
deficient regimes as the difference between the deficient and nondeficient nitrogen regimes.
In the non-deficient nitrogen regime the total soluble reduced-N
fraction in the tops was significantly lower when nitrate was the N-source
than when nitrogen was supplied as ammonium or ammonium nitrate; but it
was not significantly lower than the level in the tops of the control
plants.
In the roots no significant changes were detected.
Significant differences were not detected among sources in the
total soluble reduced-N fraction of either the tops or roots when plants
were pretreated for 5 days without nitrogen.
Significant increases occurred in the total soluble reduced-N
fraction of the tops and roots of plants grown 15 days without nitrogen
and then supplied a nitrate N-source.
No significant differences were
effected by ammonium or ammonium nitrate sources.
Highly significant differences were detected in the total
Kjeldahl-N fraction (fig. 21 and 22) of plants with respect to nitrogen
regime, N-source and plant part.
The total Kjeldahl-N concentration of
the plant tissue decreased as the plants became more and more nitrogen
deficient.
Analysis of the overall effect of N-source showed that the
control plants and plants supplied a nitrate N-source had a significantly
higher Kjeldahl-N content than the plants supplied ammonium or ammonium
nitrate N-sources. The total Kjeldahl-N concentration in the root tissue
was higher than in the tops.
In the non-deficient nitrogen regime no significant differences
were detected in either the tops or the roots.
However, when ammonium
66
ESI »
O =
E3 2ZZ2
CONTROL
N
N03
NH4N03
36
lu
z>
30
CD
CD
h
25
•ZL
<
-j
cl
>-
20
cc
q
C3>
e
o>
E
2:
ll!
CD
o
£e
h-
COMPLETE
fig. 21
MINUS N
5 DAYS
MINUS N
15 DAYS
total kjeldahl-n fraction for different
nitrogen regimes and n-sources - tops
67
EZZZZI
=
mm .
complete
nh4
C=3 « N03
es
-
nh4n03
44
w
40
CO
j?
-J
d_
>
36
28
c£
q
24
complete
minus n
5 days
minus n
15 days
fig. 22 total kjeldahl -n fraction for different
nitrogen regimes and n-sources - roots
63
was the N-source the total Kjeldahl-N content in both the tops and roots
appeared lower than in the control plants or the plants supplied ammonium
nitrate or nitrate- N-sources.
In the plants which were nitrogen deficient for 5 days the total
Kjeldahl-N in the roots exhibited no significant changes.
However, the
tops of plants supplied ammonium nitrate as the N-source were signifi­
cantly lower in total Kjeldahl-N than the control plants. There was no
significant difference in the total Kjeldahl-N fraction among the Nsources.
In both tops and roots of plants nitrogen deficient for 15 days
the total Kjeldahl-N of plants supplied an ammonium N-source signifi­
cantly decreased below the total Kjeldahl-N level in the control plants.
The same decline was observed in the tops of plants supplied an ammonium
nitrate N-source; the total Kjeldahl-N in the roots, however, was not
significantly different from the control.
When nitrate was the N-source
the total Kjeldahl-N in the tops was significantly higher than the level
in the control plants or plants supplied ammonium and ammonium nitrate
N-sources.
Changes in total Kjeldahl-N in the roots followed the same
general pattern as in the tops although the nitrate source did not ef­
fect a significant increase in total Kjeldahl-N over the control.
Data in Tables 6 and 7 show the per cent total nigrogen (total
Kjeldahl-N + nitrate-N) in the tops and roots for the different nitrogen
regimes and N-sources.
These data corraborate the findings based on the
nitrogen level in the plants and the absorption of the various N-sources
from the culture solution.
69
TABLE 6
PER CENT TOTAL NITROGEN IN DRY PLANT TISSUE—TOPS AND ROOTS
Nitrogen
Regime
Nitrogen
Source
Plant Part*
Tops
Roots
Per Cent"1"—
Complete
Minus N 5 days
Minus N 1 5 days
Control
4.33 g
4.43 c
NH.
4
3 . 4 5 ef
4.02 c
no3
3.95 fg
4.68 c
NH4N03
3.97 fg
4.78 c
Control
3 . 1 0 de
2.91 b
NH4
2 . 5 6 cd
2.71 b
no3
2 . 5 5 cd
2.93 b
nh4no3
2 . 1 3 be
2.72 b
Control
1 . 5 9 ab
2 . 0 2 ab
1.09 a
1.09 a
no3
3 . 9 2 efg
2.75 b
NH4N03
1.04 a
1 . 7 4 ab
NH4
*Nitrogen regime by nitrogen source interaction:
Tops sx = 0.213, Roots s- = 0.360.
+Values are means of 3 replications. Values followed by the same letter
within a plant part are not significantly different.
TABLE 7
MEANS OF MAIN EFFECTS EXPRESSED AS PER CENT TOTAL
NITROGEN IN DRY PLANT TISSUE—TOPS AND ROOTS
Nitrogen
Regime
Plant Part
Roots
Tops
—Per Cent*
Complete
3.84 b
4.48 c
Minus N 5 days
2.58 a
2.81 b
Minus N 15 days
2.02 a
1.90 a
*Values followed by the same letter within a given plant part are not
significantly different; Tops s- = 0.175, Roots s- = 0.020.
•
Nitrogen
Source
Plant Part
Tops
Roots
—Per
Control
3.14 b
3.13 ab
nh4
2.28 a
2.61 a
N03
3.46 b
3.48 b
nh4no3
2.37 a
3.04 ab
*Values followed with the same letter in a given plant part are not
significantly different; Tops s- = 0.123, Roots s^ = 0.207.
71
Perhaps the most outstanding aspect of the greenhouse experiment
was the large increase observed in the total Kjeldahl-N in the tops and
roots of plants which were extremely nitrogen deficient and then supplied
a nitrate N-source.
Within a 24-hour period these plants had absorbed,
reduced, and converted to protein a large amount of nitrate-N. The rate
at which assimilation was occurring and the amount of nitrate-N involved
indicates that respiration was not competing with nitrate reduction for
the NADH co-enzyme as suggested by Syrett (30).
If there was competition
between nitrate reduction and respiration, then respiration would have
had to almost cease in order to allow for such a large amount of nitrate
to be reduced.
It appears that the protein fraction is very labile in
young plants and that some portion of the total protein content in
plants serves as a storage form of nitrogen.
Part of the increase observed in the total Kjeldahl-N of defi­
cient plants which were supplied nitrate-N occurred in the total soluble
reduced-N fraction, although most of the increase occurred in the pro­
tein concentration in the plants.
Vomhof (36) suggested that the total
soluble reduced-N concentration in plant tissue was indicative of the
nutritional status of the plants.
the
He also found qualitative changes in
-amino acids in this fraction.
Evidence indicates a rapid decline in the total soluble reduced-N
approached a minimum concentration as the plants became mildly nitrogen
deficient.
A corresponding decrease in the protein occurred.
With more
severe deficiency the rate of protein degradation appeared to increase.
Thus, the extent of nitrogen deficiency was better indicated by the
protein concentration in the plant tissue than the total soluble reduced-N
fraction.
Past studies have sometimes alluded to a preference for nitrate-N
by cotton plants but it has never been clearly demonstrated.
Field ob­
servations have indicated that cotton plants recover from a nitrogen de­
ficiency more quickly when nitrate-N is supplied the plants than when any
other form of nitrogen is supplied. Gardner (6) related increases in the
nitrate concentration in the petiole of cotton plants to applications of
ammonium nitrate to nitrogen deficient plants.
The nitrate content of
the petiole was in turn highly correlated with yield of seed cotton.
Under conditions of the experiment conducted herein, cotton plants ex­
hibited a preference both in the absorption and utilization of nitrate-N.
The practical value of this information becomes apparent. The
loss of protein from young plant tissue as the plants become more and
more deficient has numerous ramifications with respect to the overall
metabolism of the plant.
All aspects of cell multiplication and plant
development leading to fruiting would be necessarily affected.
In addi­
tion, the response of extremely deficient plants to nitrate-N stresses
the necessity of selecting the proper nitrogen source in cultural prac­
tices designed to overcome nitrogen deficiency that has developed in
young cotton plants.
SUMMARY AND CONCLUSIONS
Acala 4-42 was the variety of cotton used to study the influence
of N-source on the absorption and utilization of nitrogen.
In addition,
the conditions of presence or absence of light and degree of nitrogen
deficiency were studied to determine if they influenced the assimilation
of inorganic N-sources. The plants which were used in the experiments
were grown in a culture solution in either a greenhouse or a growth
chamber.
The data used in constructing the figures seen in the text are
presented in Tables 8 through 12 in the Appendix.
The observations and conclusions based on data from these experi­
ments are given as follows:
1.
Urea and
-amino acids were absorbed by cotton plants as
intact molecules.
2.
Light regime and N-source did not independently influence
the absorption and utilization of the inorganic N-sources.
The effect
of light regime on absorption and utilization of nitrogen was more evi­
dent when ammonium was the N-source.
3.
When ammonium was the N-source the protein fraction in the
plant appeared to be rapidly degraded into a more mobile form of nitrogen.
4.
The exudation of soluble reduced-N compounds into the culture
solution was occurring concomitantly with absorption of nitrogen from the
culture solution.
5.
A preference for the absorption and utilization of the nitrate
nitrogen source was exhibited by extremely nitrogen deficient plants.
73
appendix
74
TABLE 8
PRELIMINARY GROWTH CHAMBER STUDY WITH AMMONIUM
AND NITRATE N-SOURCES—TOPS AND ROOTS
Nitrogen
Source
Time
Hours
Ammonium
Tops
Roots
Nitrogen Fractions
Total Soluble
Nitrate
Kj eldahl-N
Tops
Roots
Tops
Roots
mgm N/gm Dry Plant Tissue*
Total
Kjeldahl-N
Tops
Roots
Control
0
0.31
0.64
0.24
0.89
2.78
2.38
20.40
22.32
Ammonium
2
0.30
0.59
0.25
0.70
2.17
3.74
16.37
14.91
6
0.27
0.56
0.13
0.58
1.64
3.37
18.32
20.77
12
0.31
0.36
0.23
0.33
2.43
3.15
18.44
18.90
24
0.30
1.23
0.25
0.63
2.95
3.09
20.28
24.46
2
0.14
0.70
0.46
3.99
1.81
2.89
19.07
25.32
6
0.24
1.89
0.53
3.50
1.81
3.62
19.90
25.46
12
0.33
0.55
0.86
4.92
3.81
4.53
20.96
27.42
24
0.50
0.50
1.53
8.23
4.20
5.63
20.64
28.33
Nitrate
•Values are means of 2 replications.
TABLE 9
THE NITROGEN FRACTIONS OF PLANTS GRCWN IN THE LIGHT-DARK CYCLE
WITH VARIOUS INORGANIC N-SOURCES—TOPS AND ROOTS
Nitrogen
Fraction
Time
0.17
0.36
0.20
0.26
0.38
H
0.70
Nitrate
1.46
fcl
1.47
*"2
1.74
fc3
2.63
Total Soluble
3.67
to
Kj eldahl
5.21
fcl
2.49
fc2
4.07
fc3
4.92
*4
t
Total kjeldahl
21.54
t.
23.23
23.43
fc2
27.50
27.30
fc4
•Values are means of 3 replications.
^These values are too low; the values
t^ and t2 respectively.
Ammonium
tQ
*1
fc2
fc3
Ammonium
Tops
Roots
-J
A
0.28
0.46
0.49
0.59
0.58
0.30
0.58
0.52
0.22
0.80
3.12
3.09
4.22
5.68
4.60
25.53
21.24
25.51
25.88
26.05
Nitrogen Source
Nitrate
Ammonium Nitrate
Tops
Roots
Tops
Roots
mgm N/gm Dry Plant Tissue*—
—
0.21
0.38
0.34
0.51
0.35
0.80
2.31
1.50
1.71
2.05
3.10
5.61
4.87
6.01
4.92
21.34
25.71
25.50
28.39
27.10
0.30
0.38
0.30
0.35
:.28
0.20
1.37
1.29
2.34
1.16
4.19
3.65
3.84
5.16
4.05
20.88
28.90
24.07
26.90
23.99
0.20
0.27
0.31
0.28
0.35
1.23
1.29
1.09
1.25
0.89
2.50
3.92
3.18
3.76
3.83
20.95
20.45
20.65#
23.33
22.24
0.29
0.80
0.59
0.52
0.41
0.32
1.63
1.86
1.02
1.60
2.15
4.16
3.83
3.61
3.17
23.54
21.65
26.51
29 .49
25.08
based on means of 2 replications are 22.93 and 23.08 for
77
TABLE 10
THE NITROGEN FRACTION OF PLANTS GROWN IN THE PROLONGED LIGHT
CYCLE WITH VARIOUS INORGANIC N-SOURCES—TOPS AND ROOTS
Nitrogen Source
Nitrogen
Fraction
Ammonium
Ammonium
Ammonium
Nitrate
Nitrate
Tops
Roots
Tops
Roots
Tops
Roots
mgm N/gm Dry Plant Tissue*
T ime
0.27
0.33
0.21
0.39
0.21
0.17
0.34
0.43
0.26
0.20
0.20
0.48
0.28
0.33
0.17
0.27
0.35
0.62
0.22
0.51
0.30
0.41
0.32
0.43
1.23
0.36
0.56
0.24
0.92
0.21
1.47
0.70
3.44
1.58
2.22
0.91
0.78
0.78
1.17
0.49
0.95
1.39
2.01
0.29
2.90
1.49
2.82
1.06
2.43
1.91
3.80
1.42
1.49
1.07
3.95
3.14
3.03
1.75
3.53
4.84
3.38
1.68
3.03
4.36
5.19
5.53
3.10
2.33
2.77
3.02
4.42
3.34
21.94
29.48
18.33
26.74
23.03
32.67
25.61
23.90
28.19
44.98
26.70
26.28
18.39
27.92
24.84
30.96
23.85
22.61
19.26
31.31
26.55
31.82
25.38
32.12
fco
fcl
fc2
fc3
Nitrate
V
*3
Total Soluble
Kjeldahl
u
*1
t2
fc3
Total Kjeldahl
t
o
*1
fc2
S
*Means of 3 replications.
78
TABLE 11
THE NITROGEN FRACTION OF PLANTS GROWN IN THE PROLONGED DARK
CYCLE WITH VARIOUS INORGANIC N-SOURCES —TOPS AND ROOTS
Nitrogen Source
Nitrogen
Fraction
Ammonium
Time
0.15
0.28
0.16
0.23
0.15
0.34
0.28
0.32
0.25
0.34
0.17
0.38
0.24
0.77
0.36
0.41
0.65
0.57
0.27
0.51
0.26
0.26
0.25
0.40
0.75
0.31
0.65
0.23
0.60
0.27
2.16
0.99
2.01
1.01
1.32
0.58
1.82
0.68
1.37
2.08
0.76
0.99
3.10
0.80
2.71
1.69
1.77
1.20
t
o
1.90
2.25
1.89
2.85
1.65
2.03
t
1
4.24
2.59
3.72
3.05
3.62
2.27
3.30
7.11
6.23
5.77
8.02
13.49
4.17
3.55
3.67
3.93
3.52
3.45
22.48
25.81
23.13
28.17
19.95
30.37
25.81
17.57
23.03
28.75
23.58
32.60
19.58
34.36
33.25
24.27
23.43
29.67
27.80
28.32
26.85
29.71
22.19
32.34
t
o
fc2
S
Nitrate
to
*1
*3
Total Soluble
Kjeldahl
fc2
fc3
Total Kjeldahl
Ammonium
Ammonium
Nitrate
Nitrate
Tops
Roots
Tops
Roots
Tops
Roots
— mgm N/gm Dry Plant Tissue*
**
*1
*3
•Values are means of 3 replications.
TABLE 12
THE NITROGEN FRACTION OF PLANTS GROWN WITH INORGANIC N-SOURCES
FOR DIFFERENT NITROGEN REGIMES—TOPS AND ROOTS
Nitrogen Fractions
Total Soluble
Nitrate
Kjeldahl-N
Tops
Roots
Tops
Roots
mgm N/gm Dry Plant Tissue*
Total
Kjeldahl-N
Tops
Roots
Nitrogen
Regime
Nitrogen
Source
Ammonium
Tops
Roots
Complete
Control
0.92
0.62
8.17
3.18
10.22
10.07
35.05
41.15
NH,
1.09
1.10
5.87
3.24
12.01
10.41
28.86
37.14
N0„
0.53
0.89
8.08
6.58
8.14
10.60
31.41
41.21
nh4no3
1.20
0.70
7.12
4.37
12.66
11.75
32.32
42.40
Control
0.39
0.33
3.27
0.73
6.58
4.57
27.60
28.59
NH,
0.51
0.72
2.98
0.77
7.27
7.01
20.76
-26.43
NO.
0.33
0.37
2.41
2.24
5.09
6.43
22.76
26.81
nh4no3
0.48
0.54
2.26
0.27
6.85
7.59
19.10
26.85
0.39
0.42
1.33
1.89
4.96
5.00
18.65
18.27
NH.
0.31
0.42
0.35
0.11
4.19
4.12
10.92
10.83
NO.
0.73
0.51
6.60
3.87
9.90
9.10
32.67
23.67
NH.NO.
4 3
0.23
0.55
0.28
0.99
3.87
3.92
10.11
16.37
Minus N 5 days
Minus N 15 days Control
*Values are means of 3 replications.
REFERENCES
Ambadasrao, P., 1960. Efficacy of different sources of nitrogen,
i.e., urea and ammonium nitrate compared to ammonium sulfate,
with and without phosphoric acid, in increasing the yield of
cotton on B.C. soils under Tungabhadra Project (Agr. Res. Stn.
Dhadesugar, India). Mysore Agr. J. 35:161-166.
Barker, A. B., K. J. Volk, and W. A. Jackson, 1966. Root environment
acidity as a regulatory factor in ammonium assimilation by the
bean plant. Plant Physiology 41:1193-1196.
Bremmer, J. M., 1965. Methods of soil analysis; Part 2—Chemical and
microbiological properties. Agronomy 9:1149-1345.
Dastur, R. H., and M. J. Dawson, 1962. Growth and yields of Egyptian
Cotton under irrigated conditions in Mysore. II—Nitrogen and
mineral composition of Egyptian Cotton in Mysore. Indian J. Agr.
Sci. 32:167-177.
Dejaegere, Robert, 1964. The effects of the nature of the nitrogen
supply on the growth of cotton plants. Ann. Physiol. Vegetale.
Univ. Bruxelles 9:12-16.
Gardner, Bryant R., 1963. A study of factors influencing the nitro­
gen fertilization of Acala Cotton. Ph.D. Dissertation University
of Arizona.
Ghosh, B. P. and R. J. Burris, 1950. Utilization of nitrogenous com­
pounds by plants. Soil Sci. 70:187-203.
Hall, E. E., and S. J. Watson, 1932. Research work with cotton (Pee
Dee Experiment Station), S. Car. Agr. Expt. Stn. 45th Ann. Rept.
pp. 110-115.
Hattori, A., 1957. Studies on the metabolism of urea and other nitro­
genous compounds in Chlorella ellipsoidea. I. Assimilation of
urea and other nitrogenous compounds by nitrogen-starved cells.
J. of Biochem. 44:253-273.
Hoagland, 0. R., and 0. I. Arnon, 1950. The water-culture method for
growing plants without soil. Calif. Agr. Expt. Stn. Cir. 347
(revised).
Holley, K. T., T. A. Pickett, and T. G. Dulin, 1931. A study of
ammonia and nitrate nitrogen for cotton. I. Influence on ab­
sorption of other elements. Georgia Expt. Stn. Bull. 169:1-15.
80
81
12.
Holley, K. T., T. G. Dulin and T. A. Pickett, 1934. A study of
ammonium and nitrate nitrogen for cotton. II. Influence on
fruiting and some organic constituents. Georgia Expt. Stn. Bull.
182:1-30.
13.
Holley, K. T., and T. G. Dulin, 1935. A study of ammonia and nitrate
for cotton. III. Influence of the nitrogen concentration in the
nutrient medium. Georgia Expt. Stn. Bull. 197:1-14.
14.
Holley, K. T., and T. G. Dulin, 1943. A study of ammonia and nitrate
for cotton. V. Influence of variety. Georgia Expt. Stn. Bull.
229: 2-8.
Ivanova, V. S., 1934. Utilization of ammonia nitrogen by cotton.
Lenin Acad. Agr. Sci. Gedroiz Inst. Fertilizers Agro-Soil Sci.
No. 3: 77-103.
15.
16. Kirby, E. A., and K. Mengel, 1967. Ionic balance in different tis­
sues of the tomato plant in relation to nitrate, urea, or
ammonium nutrition. Plant Physiology 42:6-14.
17.
Koren 1 Kov, D.A., 1963. Agrochemical evaluation of urea fertilizers.
Zemledelie 10:30-34.
18.
Kuykendall, Roy, 1933-1935. Twelve years' results with nitrogen
fertilizers on cotton and corn. Proc. 34th, 35th, and 36th Ann.
Conventions of Assoc. Southern Agr. Workers, pp. 75-76.
19.
Lycklama, J. D., 1963. The absorption of ammonium and nitrate by
perennial rye-grass. Acta Botanica Norlandia 12:361-423.
20.
Maskell, E. J., and T. G. Mason, 1929. Studies on the transport of
nitrogenous substances in the cotton plant. I. Preliminary
observations on the downward transport of nitrogen in the stem.
Ann. of Bot. 43:205-231.
21.
Maskell, E. J., and T. G. Mason, 1929. Studies on the transport of
nitrogenous substances in the cotton plant. II. Observations
on concentration gradients. Ann. of Bot. 43:615-650.
22.
Naftel, James A., 1931. The absorption of ammonium and nitrate
nitrogen by various plants at different stages of growth. J.
Am. Soc. Agron. 23:142-158.
23.
Neirinckx, Louis J. A., 1964. Influence of the nutrient medium
(ration of anions/cations, and total dose) on the mineral com­
position of the cotton plant. Ann. Physiol. Vegetale Univ.
Bruxelles 9: 57-59.
24.
Paden, W. R., and W. H. Garman, 1947. Yield and composition'of cot­
ton and Kobe lespedeza grown at different pH levels. Soil Sci.
AM. Proc. Il:309-31b.
82
25.
Pershin, G. P., 1950. The relation of seedlings of cotton plants to
the sources of nitrogen and aeration of the nutritive solution.
Doklady Akad. Nauk. Uzbek. S.S.R. 12:38-40.
26.
Phillis, E., and T. G. Mason, 1942. Studies on the partition of the
mineral elements in the cotton plant. II. On the diurnal vari­
ations in the mineral content of the leaf of the cotton plant.
Ann. of Bot. 6:437-442.
27.
Phillis, E., and T. G. Mason, 1942. Studies on the partition of the
mineral elements in the cotton plant. 111. Mainly concerning
nitrogen. Ann. of Bot. 6:469-485.
28.
Rauschkolb, R. S., and T. C. Tucker, 1966. Adaptation of Macro
Kjeldahl Digestion Racks for use with Micro Flasks. ChemistAnalyst. 55:21.
29.
Steel, Robert G. D., and J. H. Torrie, 1960. Principles and proce­
dures of Statistics. McGraw-Hill Book Co., Inc. New York, p.
108.
30.
Syrett, P. J., 1956. The assimilation of ammonia and nitrate by
nitrogen starved cells of Chlorella vulgaris. II. Assimilation
of large quantities of nitrogen. Physiol. Plant. 9:19-27.
31.
Tiedjens, Victor A., 1932. Growing cotton and other crops with am­
monium nitrogen. Science 75:648-651.
32.
Tokuoka, Matuo, and Suisen Dyo, 1938. Fertilization of the cotton
plant. II. The actions of nitrogen fertilizers. J. Soc. Trop.
Agr. Taihoku Imp. Univ. 10:16-20.
33.
van Schoor, Geramine H. S., 1962. The mineral composition of the
cotton plant as related to its mineral supply. Ann. Physiol.
Vegetele. Univ. Bruxelles 7:328-331.
34.
Virtanen, Artturi I., and Hilkka Linkola, 1946. Organic nitrogen
compounds in nitrogen nutrition for higher plants. Nature
158:515-517.
35.
Vines, H. N., and R. T. Wedding, 1960. Some effects of ammonia on
plant metabolism and a possible mechanism for ammonia toxicity.
Plant Physiology 35:820-823.
36.
Vomhof, Daniel W., 1967. Inter-relationships between carbohydrate
and nitrogen availability and boll shedding in Gossypium. Ph.D.
Dissertation. Univ. of Arizona.
37.
Weissman, Gerard S., 1964. Effect of ammonia and nitrate to nutri­
tion on protein level and exudate composition. Plant Physiology
39:947-951.
Willis, L. G., and E. A. Davis, 1928. The toxicity to cotton seed­
lings of high concentration of soluble nitrogenous fertilizers.
N. Car. Agr. Expt. Stn. Tech. Bull. 30:1-12.
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