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Incorporation of N15-hydroxylamine into the proteins of wheat seedlings
Life Sciences No . 3, pp . 184-189, 1963 . Pergamon Press, Inc . Printed in the United States .
INCORPORATION OF N 15 -HYDROXYIAMINE INTO THE PROTEINS OF WHEAT SEEDLINGS W . L . Kretovich~ A, A . Bundel~ N . V . Borovikova A, N . Bach Institute of Biochemistry Academy of Sciences of the USSR (Received 24 January 1963) DATA presented elsewhere
(1~2) have shown that hydroxylamine is an inter-
mediate formed in the process of nitrate assimilation by the plant .
The
introduction of hydroxylamine at various concentrations into living plants or plant homogenates resulted in the accumulation of glutamic acid s
serine, aspartic acid s glutamine and asparagine .
The effect of
hydroxylamine on the growth and development of wheat seedlings was also studied
(3) .
It was found that after the absorption of hydroxylamine
through the roots into wheat seedlings or after the introduction of solutions of hydroxylamine hydrochloride
(0 .001 M) or equimolar hydroxyl-
amine hydrochloride and rz-ketoglutaric acid tion~
(0 .01 M) by vacuum infiltra-
the seedlings planted into the soil underwent a normal growth and
development,
On the basis of the data obtained
(4) the conclusion was
drawn that hydroxylamine actually was an intermediate in the reduction of nitrate to ammonia, The present paper describes a study of the incorporation of N15hydroxylamine nitrogen into the proteins of wheat seedlings . Expe riments The preparation of N 15 -hydroxylamine contained 10,7 atom per cent N 15 and was free from nitrates, nitrites and ammonia, Seeds of the summer wheat, variety "Moscovka"were germinated .
184
On
x1 5_amxox~m~ n~ca~xPORaT~ n~ro
xo . 3
WHPAT
s~rn~c ~xoT~Ixs
the fourth day after the beginning of moiatening~ caryopses were detached and the green seedlings were set out on paraffin-soaked gauze plates stretched on a frame made of glass rode .
The roots of the seedlings were
let through orifices in plates placed over vessels of appropriate shape which contained Rnop' solution in which Ca(NOg)2 was replaced by a corresponding amount of CaS04 . each plate .
Seventy to eighty sprouts were arranged on
During the first days of the experiment Rnop' solution was
diluted with water (1 ;4) . In one experimental aeries
N 15 -hydroxylamine or N 15 -oxime solutions
(pH 5 .8 - 5 .9) were introduced into living seedlings through the rootsyatem~ in another series they were introduced by vacuum infiltration . We chose the highest concentrations of hydroxylamine and oxime which did not unfavorably affect the seedlings under the experimental conditions described .
This gave ue the possibility to introduce into the
plants a high enough dose of N 15 -hydroxylamine to determine N 15 content in the plant proteins by a mass-spectrometer .
This concentration was
0 .001 M of hydroxylamine and 0,01 M of oxime . The solutions to be introduced into the plants were prepared shortly before the experiments . sodium bicarbonate .
N 15H20H " HC1 was neutralized to pH 5 .8 - 5 .9 with
To obtain the oxime
0 .01 M N 15H20H " HC1 solution was
mixed with an equal amount of (x-ketoglutaric acid solution (0 .02 M) two hours prior to the experiment and the solution was brought to the same pH . Introduction of N 15 H~OH " HC1 and N 15 -oxime Solutions Through the Boot System From the sixth day of moistening of the seeds
the plate with the
seedlings was transferred daily from the glasses with Bnop's mixture without nitrogen to simflar glasses containing freshly prepared test solubona ; the plates were placed in such a way that the roots were completely immersed in the solutiona~ while the leaves remained under natural light . The plates were left in this way for one hour s glasses with Rnop'
solution lacking nitrogen .
then again placed on the The experiment was con-
i85
i86
N1 5_gyIaROäSCIAI~ INCORPORATED II~llb WHEAT SEEDLING PROTEINS
tinned for 30 days .
No .
In control experiments the roots of the plant remained
immersed in Rnop' solution without nitrogen during the time of the experiment . At the end of the experiment,
the plants were weighed,
counted,
frozen in dry ice and alcohol, andlyophylized . Total nitrogen in the dry material was determined by Kjeldal method and protein nitrogen by method of Bannstein .
To determine the N 15 content
ammonia was distilled into a receiver containing 0 .02 N H2 S04 . samples, which contained N 15 in the form of
These
(N 15~)2504, were acidified
with several drops of 0 .02 N H 2 S04 , concentrated to a volume of 3 - 5 ml, depending upon nitrogen content in the samples, converted to gaseous nitrogen by Rittenberg's method (5) and then analyzed by mass spectrometry for the content of N15 .
The determination was carried out three or four
times in separate samples . Introduction of Hydrox~+lamine an d Oxime into Wheat Seedlings by Means of Vacuum Infiltration Seedünge devoid of caryopaea were grown by the method described In order to carry out vacuum infiltration,
above .
the whole plate with
the seedlings was placed into a vessel containing the solution to be in filtrated .
The vessel with both the solution and the plants was placed
in a vacuum desiccator and vacuum infiltration was performed by the usual method .
The plants and the plates then were washed with distilled water
and again placed on the glasses with Knop' solution lacking nitrogen . The vacuum infiltration procedure was performed three times with the oxime
(0 .01 M) and five times with hydroxylamine
(0 .001 M), since the
hydroxylamine solution was 10 times weaker than that of the oxime .
In
the control experiment seedlings of the same group s also devoid of caryopaea, were exposed five times to vacuum infiltration with water .
To
elucidate the effect of vacuum infiltration itself upon the seedlinga~ a parallel, control experiment without vacuum infiltration was performed on the seedlings of the same net of seeds devoid of caryopaea, moistened on
3
Nl5_gyDROXYLA~s INCORPOIiATED IIiTO WSEAT S~EDLING PROTEINS
No . 3
the same day as the experimental ones . In the aeries with the introduction of the oxime vacuum infiltration
after the third
the plants were allowed to grow on Rnop's mixture
without nitrogen to the end of the experiment and then analyzed .
Vacuum
infiltration was carried out on the sixth s ninths sixteenths twenty-fourth and twenty-eighth day after moistening of the seeds . Results IIpon introduction of N 15-hydroxylamine and N 15-oxime solutions through the roots of wheat plants considerable amounts of heavy nitrogen were incorporated into the plants
(Table 1) .
The per cent of N15 in the
protein of these sprouts was also increasing . The results of two experiments performed at different times are in good agreement . The dry weight of the plants averaged 19 .8 mg in the controls mg after treatment with oxime
19 .3
11,4 mg after treatment with hydroxylamine,
The lowered weight produced by hydroxylamine seems to be related to the inhibition of photosynthetic processes and to the accumulation of carbohydrate .
The calculated content of protein nitrogen in mg per plant :
0 .30 in the control s 0 .46 after oxime treatment and 0,38 after hydroxylamine treatment . After introduction of N 15 -hydroxylamine and N15 -oxime solutions into wheat seedlings by vacuum infiltration
heavy nitrogen was incorporated
into the protein of the seedlings ; the incorporation was particularly high after treatment with the oxime . also considerably increased,
The mean weight of each plant
It is interesting to note that the mean
weight of each plant in the group without vacuum infiltration was 95 mg r i .e, considerably lower than that in the control with vacuum infiltration of water (115 mg) .
187
1.88
xi5-
n~c
nrro w~ sg~Ln~G PRO~sIxs
No . 3
TABLE 1 N15 Incorporation into Proteins of Wheat Seedlings upon the Application of N 1 ~I2 0H'RC1 and N15 -oxime Solutions through the Roots
NN of Exper
1.
2.
Conditions of Growth
Nitrogen (X per airdry substance) protein total
N 15 content in protein nitrogen atom Z atom X excess N15
Control without nitrogen
1,65
1,90
0,38
N 15 -oxime
2,59
3,23
3,58
3 .20
N 15ü 2 0H " ~1
2,11
3,06
2 .07
1,69
Control without nitrogen
1 .64
1,72
0 .38
N 15 -oxime
2,37
3 .14
3 .67
3 .29
N 15 H2 0H " HC1
2,43
3 .65
1,58
1 .20
TABLE 2 N15 Incorporation into Proteins of Wheat Seedlings upon Vacuum Infiltration of N 15 H20H'üCl and N 15 -oxime Solutions
Conditions of Growth
Mean Weight of Planta at end of Experiment (mg)
Nitrogen per 10 Planta (mg) protein total
N 15 Content in Protein Nitrogen atom X atom X excess
Control without nitrogen
115
2,40
3 .22
0 .38
N15 H20H " EC1
128
2,48
3 .46
1 .02
0 .64
N 15 -oxime
138
2 .90
3 .86
1,14
0,76
~15_~o~orr~m~
xo . 3
n~coxP~A~ nrro
zas
W~ ~Lta~
Conclusions 1,
Upon daily introduction of N 15H20H " i~Cl
of pI-ketoglutaric acid wheat seedlings
(0 .001 M) and N 15-oxime
(0 .01 M) solutions through the root system of
the atom per cent N 15 in proteins of the plants was 3 .58
and 3 .67 after N15 -oxime treatment and 2 .07 and 1 .58 after N 15H20H " HC1 treatment (atom per cent N 15 in the hydroxylamine used was 10 .7) . 2,
In other experiments the N 15 -compounds were introduced into
seedlings through vacuum infiltration .
In the case of N15 -oxime (0 .01 M)
the vacuum infiltration was repeated three timea~ while N 15H20H " HC1 (0 .001 M) was repeated five times .
After 30 days the N 15 content of
protein in the plants after treatment with N 15H20H " i~Cl was 1,02 atom and after treatment with the isotopic oxime was 1,14 atom 3.
x
x.
The data obtained by two different methods show the nitrogen of
hydroxylamine as the hydrochloride or the oxime can enter into the reacbone of protein synthesis in wheat seedlings .
These results would sug-
gent that in plants hydroxylamine is an intermediate in the reduction of nitrate to a®onia . Referencea 1.
W, L . RRSTOVICH~ A . A . BUNDEL~ M, R . FRASHBRI and N . V . BOROVIROVA~ Dokladi Akad, nauk SSSR 1~
2.
~
414
(1958) .
W . L . RßETOVICH~ A . A . BUNDSL~ M . R, FRASHSRI and N . V . BOROVIROVA~ Fiaiologia rasten, 8~ issue 3~ 261
4.
(1958),
W . L . RRSTWICH~ A . A . BUNDSL~ M . R, FRASHSRI and N . V . BOROVIROVA~ Zhurn, obahch . biol,
3.
1065
(1960) .
W . L . I~ETOVICH~ Biochemistry of Autotrophic Assimilation of Nitrogen . 16th Bach Lecture s Akad, nauk SSSR~ Moscow (1961) .
5.
D . RI1I~SRG~ Obtaining and Determination of Labeled Atoms . tion of Papers
Collec-
Foreign Literature Publishing House s Moscow (1948) .
189
INCORPORATION OF N 15 -HYDROXYIAMINE INTO THE PROTEINS OF WHEAT SEEDLINGS W . L . Kretovich~ A, A . Bundel~ N . V . Borovikova A, N . Bach Institute of Biochemistry Academy of Sciences of the USSR (Received 24 January 1963) DATA presented elsewhere
(1~2) have shown that hydroxylamine is an inter-
mediate formed in the process of nitrate assimilation by the plant .
The
introduction of hydroxylamine at various concentrations into living plants or plant homogenates resulted in the accumulation of glutamic acid s
serine, aspartic acid s glutamine and asparagine .
The effect of
hydroxylamine on the growth and development of wheat seedlings was also studied
(3) .
It was found that after the absorption of hydroxylamine
through the roots into wheat seedlings or after the introduction of solutions of hydroxylamine hydrochloride
(0 .001 M) or equimolar hydroxyl-
amine hydrochloride and rz-ketoglutaric acid tion~
(0 .01 M) by vacuum infiltra-
the seedlings planted into the soil underwent a normal growth and
development,
On the basis of the data obtained
(4) the conclusion was
drawn that hydroxylamine actually was an intermediate in the reduction of nitrate to ammonia, The present paper describes a study of the incorporation of N15hydroxylamine nitrogen into the proteins of wheat seedlings . Expe riments The preparation of N 15 -hydroxylamine contained 10,7 atom per cent N 15 and was free from nitrates, nitrites and ammonia, Seeds of the summer wheat, variety "Moscovka"were germinated .
184
On
x1 5_amxox~m~ n~ca~xPORaT~ n~ro
xo . 3
WHPAT
s~rn~c ~xoT~Ixs
the fourth day after the beginning of moiatening~ caryopses were detached and the green seedlings were set out on paraffin-soaked gauze plates stretched on a frame made of glass rode .
The roots of the seedlings were
let through orifices in plates placed over vessels of appropriate shape which contained Rnop' solution in which Ca(NOg)2 was replaced by a corresponding amount of CaS04 . each plate .
Seventy to eighty sprouts were arranged on
During the first days of the experiment Rnop' solution was
diluted with water (1 ;4) . In one experimental aeries
N 15 -hydroxylamine or N 15 -oxime solutions
(pH 5 .8 - 5 .9) were introduced into living seedlings through the rootsyatem~ in another series they were introduced by vacuum infiltration . We chose the highest concentrations of hydroxylamine and oxime which did not unfavorably affect the seedlings under the experimental conditions described .
This gave ue the possibility to introduce into the
plants a high enough dose of N 15 -hydroxylamine to determine N 15 content in the plant proteins by a mass-spectrometer .
This concentration was
0 .001 M of hydroxylamine and 0,01 M of oxime . The solutions to be introduced into the plants were prepared shortly before the experiments . sodium bicarbonate .
N 15H20H " HC1 was neutralized to pH 5 .8 - 5 .9 with
To obtain the oxime
0 .01 M N 15H20H " HC1 solution was
mixed with an equal amount of (x-ketoglutaric acid solution (0 .02 M) two hours prior to the experiment and the solution was brought to the same pH . Introduction of N 15 H~OH " HC1 and N 15 -oxime Solutions Through the Boot System From the sixth day of moistening of the seeds
the plate with the
seedlings was transferred daily from the glasses with Bnop's mixture without nitrogen to simflar glasses containing freshly prepared test solubona ; the plates were placed in such a way that the roots were completely immersed in the solutiona~ while the leaves remained under natural light . The plates were left in this way for one hour s glasses with Rnop'
solution lacking nitrogen .
then again placed on the The experiment was con-
i85
i86
N1 5_gyIaROäSCIAI~ INCORPORATED II~llb WHEAT SEEDLING PROTEINS
tinned for 30 days .
No .
In control experiments the roots of the plant remained
immersed in Rnop' solution without nitrogen during the time of the experiment . At the end of the experiment,
the plants were weighed,
counted,
frozen in dry ice and alcohol, andlyophylized . Total nitrogen in the dry material was determined by Kjeldal method and protein nitrogen by method of Bannstein .
To determine the N 15 content
ammonia was distilled into a receiver containing 0 .02 N H2 S04 . samples, which contained N 15 in the form of
These
(N 15~)2504, were acidified
with several drops of 0 .02 N H 2 S04 , concentrated to a volume of 3 - 5 ml, depending upon nitrogen content in the samples, converted to gaseous nitrogen by Rittenberg's method (5) and then analyzed by mass spectrometry for the content of N15 .
The determination was carried out three or four
times in separate samples . Introduction of Hydrox~+lamine an d Oxime into Wheat Seedlings by Means of Vacuum Infiltration Seedünge devoid of caryopaea were grown by the method described In order to carry out vacuum infiltration,
above .
the whole plate with
the seedlings was placed into a vessel containing the solution to be in filtrated .
The vessel with both the solution and the plants was placed
in a vacuum desiccator and vacuum infiltration was performed by the usual method .
The plants and the plates then were washed with distilled water
and again placed on the glasses with Knop' solution lacking nitrogen . The vacuum infiltration procedure was performed three times with the oxime
(0 .01 M) and five times with hydroxylamine
(0 .001 M), since the
hydroxylamine solution was 10 times weaker than that of the oxime .
In
the control experiment seedlings of the same group s also devoid of caryopaea, were exposed five times to vacuum infiltration with water .
To
elucidate the effect of vacuum infiltration itself upon the seedlinga~ a parallel, control experiment without vacuum infiltration was performed on the seedlings of the same net of seeds devoid of caryopaea, moistened on
3
Nl5_gyDROXYLA~s INCORPOIiATED IIiTO WSEAT S~EDLING PROTEINS
No . 3
the same day as the experimental ones . In the aeries with the introduction of the oxime vacuum infiltration
after the third
the plants were allowed to grow on Rnop's mixture
without nitrogen to the end of the experiment and then analyzed .
Vacuum
infiltration was carried out on the sixth s ninths sixteenths twenty-fourth and twenty-eighth day after moistening of the seeds . Results IIpon introduction of N 15-hydroxylamine and N 15-oxime solutions through the roots of wheat plants considerable amounts of heavy nitrogen were incorporated into the plants
(Table 1) .
The per cent of N15 in the
protein of these sprouts was also increasing . The results of two experiments performed at different times are in good agreement . The dry weight of the plants averaged 19 .8 mg in the controls mg after treatment with oxime
19 .3
11,4 mg after treatment with hydroxylamine,
The lowered weight produced by hydroxylamine seems to be related to the inhibition of photosynthetic processes and to the accumulation of carbohydrate .
The calculated content of protein nitrogen in mg per plant :
0 .30 in the control s 0 .46 after oxime treatment and 0,38 after hydroxylamine treatment . After introduction of N 15 -hydroxylamine and N15 -oxime solutions into wheat seedlings by vacuum infiltration
heavy nitrogen was incorporated
into the protein of the seedlings ; the incorporation was particularly high after treatment with the oxime . also considerably increased,
The mean weight of each plant
It is interesting to note that the mean
weight of each plant in the group without vacuum infiltration was 95 mg r i .e, considerably lower than that in the control with vacuum infiltration of water (115 mg) .
187
1.88
xi5-
n~c
nrro w~ sg~Ln~G PRO~sIxs
No . 3
TABLE 1 N15 Incorporation into Proteins of Wheat Seedlings upon the Application of N 1 ~I2 0H'RC1 and N15 -oxime Solutions through the Roots
NN of Exper
1.
2.
Conditions of Growth
Nitrogen (X per airdry substance) protein total
N 15 content in protein nitrogen atom Z atom X excess N15
Control without nitrogen
1,65
1,90
0,38
N 15 -oxime
2,59
3,23
3,58
3 .20
N 15ü 2 0H " ~1
2,11
3,06
2 .07
1,69
Control without nitrogen
1 .64
1,72
0 .38
N 15 -oxime
2,37
3 .14
3 .67
3 .29
N 15 H2 0H " HC1
2,43
3 .65
1,58
1 .20
TABLE 2 N15 Incorporation into Proteins of Wheat Seedlings upon Vacuum Infiltration of N 15 H20H'üCl and N 15 -oxime Solutions
Conditions of Growth
Mean Weight of Planta at end of Experiment (mg)
Nitrogen per 10 Planta (mg) protein total
N 15 Content in Protein Nitrogen atom X atom X excess
Control without nitrogen
115
2,40
3 .22
0 .38
N15 H20H " EC1
128
2,48
3 .46
1 .02
0 .64
N 15 -oxime
138
2 .90
3 .86
1,14
0,76
~15_~o~orr~m~
xo . 3
n~coxP~A~ nrro
zas
W~ ~Lta~
Conclusions 1,
Upon daily introduction of N 15H20H " i~Cl
of pI-ketoglutaric acid wheat seedlings
(0 .001 M) and N 15-oxime
(0 .01 M) solutions through the root system of
the atom per cent N 15 in proteins of the plants was 3 .58
and 3 .67 after N15 -oxime treatment and 2 .07 and 1 .58 after N 15H20H " HC1 treatment (atom per cent N 15 in the hydroxylamine used was 10 .7) . 2,
In other experiments the N 15 -compounds were introduced into
seedlings through vacuum infiltration .
In the case of N15 -oxime (0 .01 M)
the vacuum infiltration was repeated three timea~ while N 15H20H " HC1 (0 .001 M) was repeated five times .
After 30 days the N 15 content of
protein in the plants after treatment with N 15H20H " i~Cl was 1,02 atom and after treatment with the isotopic oxime was 1,14 atom 3.
x
x.
The data obtained by two different methods show the nitrogen of
hydroxylamine as the hydrochloride or the oxime can enter into the reacbone of protein synthesis in wheat seedlings .
These results would sug-
gent that in plants hydroxylamine is an intermediate in the reduction of nitrate to a®onia . Referencea 1.
W, L . RRSTOVICH~ A . A . BUNDEL~ M, R . FRASHBRI and N . V . BOROVIROVA~ Dokladi Akad, nauk SSSR 1~
2.
~
414
(1958) .
W . L . RßETOVICH~ A . A . BUNDSL~ M . R, FRASHSRI and N . V . BOROVIROVA~ Fiaiologia rasten, 8~ issue 3~ 261
4.
(1958),
W . L . RRSTWICH~ A . A . BUNDSL~ M . R, FRASHSRI and N . V . BOROVIROVA~ Zhurn, obahch . biol,
3.
1065
(1960) .
W . L . I~ETOVICH~ Biochemistry of Autotrophic Assimilation of Nitrogen . 16th Bach Lecture s Akad, nauk SSSR~ Moscow (1961) .
5.
D . RI1I~SRG~ Obtaining and Determination of Labeled Atoms . tion of Papers
Collec-
Foreign Literature Publishing House s Moscow (1948) .
189