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Metabolism of radioactive asparagine in wheat leaves and Lupinus angustifolius seedlings
Metabolism of Radioactive Asparagine in Wheat Leaves and Lupinus angustifolius Seedlings’ Charles Donald Nelson? Gleb Krotkov and Guilford Bevil Reed From the Departments of Biology
and Bacteriology,
Queen’s liniversily,
Kingston,
Ontario, Canada Received December 3, 1952 INTRODUCTION
The wide distribution and high concentration of asparagine in higher plants provide ample evidence of its physiological importance. Unfortunately, our present picture of its role has been derived mainly from the results of nitrogen analysis; and experimental data pertaining to the metabolism of its carbon chain, with a few notable exceptions (I), are still wanting. It has been repeatedly observed that, in the early stages of seed germination, asparagine accumulates irrespective whether seedlings are in light or in darkness. Its disappearance in later stages occurs only in light. From this observation it has been concluded that young seedlings can produce, but not utilize, asparagine, and that utilization of asparagine can take place only in light but not in darkness. It has been felt by the present authors that metabolism of asparagine carbon chain could be conveniently studied if CY4-labeledasparagine were introduced into plants. Moreover, with the help of such an asparagine one can also examine the effect of age and light on the utilization of this compound in plants. The experiments described below were performed with these objects in mind. MATERIALS Wheat leaves (Cornell 595) and blue lupine seedlings (Lupinus angustifolius) were used as experimental material. Both were grown in flats, containing loamy soil in the case of wheat, and vermiculite in the case of lupine. Flats, kept in a special room at 22 f 5”C., were under fluorescent lights for 16 hr. daily. First leaves of wheat were used when they were 14 days old. At this age they 1 Aided by a grant from the National Research Council of Canada. 2 Holder of an Ontario Research Council scholarship. 218
METABOLIS,M
OF RADIO.4CTIVE
219
.4SP,4RAGINE
hat1 reached their full size, and carried on photosynthesis (2). Lupine seedlings Tvere used at diflerent ages. It was shown previously that in blue lupine the photosynt.hetic meirhanism is already developed in 8-dav-old seedings (3). Preparation
of Radioactive
Asparaginc
Radioactive asparagine was produced by the lupiue seedings, which were placed in a desiccat,or with about 3 millicuries (mc.) of C’WQ and illuminated 16 hr. daily for 6 days (4). Following its isolation from the seedlings, asgaragine was recrystalieed aud its purity was checked chromatographically. Of its tot.:11activity, 1.6% was found to be in aspart,ic acid. A sample of this asparagine was dissolved in water, and three aliquots of it, w(Jrc degraded with uinhydrin to liberate a-carboxyl as carbon dioxide. Degradation was carried out under the conditions described by Moore and Stein (5). From each aliquot three plates were prepared and counted with a thin-window Geigerhliillrr t,uhe. The results are given in Table I. TABLE Radioactivity
of Asparagine
I
After Reaction with Xinhydrin of Moore and Stein (6)
Sk%tlple
Activity counlsjmin.
Before degradation After degradation Aliquot 1 Aliquot 2 Aliquot 3
718 ZlZ 4” 292 f 333 f 346 f
a Standard deviation of individual
6 15 16
IJnder the Conditions Per cent of activity lost
0 59 54 52
countings.
It is clear from these data that asparagine fined to the a-carboxyl. This lack of uniform whole chain of asparagine is surprising, since C1W2 xft.er 6 days of photosynthesis. Under euamplc, is lab&d uniformiv within 1 hr.
had about 55y0 of its activity COIIdistribution of Cl1 throughout the t,his ssparagine was produced from comparable conditions glucose, for
EXPERIMENTAL
A 0.05 111solution of radioactive asparagine was vacuum infiltrated into wheat leaves and lupine seedlings (6). These were placed in dark respiration chambers, kept at 20 f 2”C., and a stream of COz-free air was drawn over them. CO2 respired was collected in 0.05 N NaOH and determined by titration. Its specific activity wm determined following plating as BaC03. After three 4-hr. periods of respiration, or a total of 12 hr., plants were ext,rarted under a reflux condenser for 12 hr. with boiling 85 % ethanol and the extracts were made to volume. Three aliquots of each alcoholic
220
NELSON,
KROTKOV
AND
REED
I 8
4
100
IO
-O------O-specific 0
I 0
activity I
I 4
Tie
in hours
88
0
I2 I2
FIQ. 1. A comparison of respiration of lupine seedlings infiltrated with water or radioactive asparagine. The dotted line indicates specific activity CL402 derived from radioactive asparagine.
either of the
extract and alcohol-insoluble cell residue were completely oxidized by a wet-combustion method (7), and the CO%produced from each aliquot was plated in triplicate and counted as BaCOo.
_
~~-__--~
~... courr(s,mirr.,fracti~,~
ll~hed
14 &!JS soluble insoluble
113lliBh.,
Old.
E.,-jY,t.
f
71,620 .5,95s 1,52J1
f f f
79,099
z?z 6,277
Ih3~~irocl
io.094 15.322 2,009
* * f
3,048 2.238 111
Tot:tl
87,425
of;
3,iHX
,2looht,I Alcohol Itt*nIGrrcl
‘rc,t :11 r,
r,,rpin.c
seedlings.
s da\ s old
Expt.
Alcohol Alcohol Respired
Ercpt.
Alcohol Alcohol Respired
649 , 9’)O .k i6,630 . 86,810 zt 32,184 0;li4+ 240
-,
soluble insoluble
~~~... __.. ~~~~~~ :SO da?.8 old Alcobol soluble Alcohol insoluble Respired Tot al
1 746.274 I j 568,887 1 96,508 1 13,921
f
83,115
f f It
76,342 21,154 365
i
679,316
zk 711,220
324,086 66,058 8,6X!
f 42,979 zk 29,659 f z20
1 398,796
~,c~>ino seedZings. Ezpt. 4 4 da\-Y oh? Alcohol soluble Alcohol insoluble Respired ToM Alcohol Alcohol Respired
soluble insoluble
Total 32 days
old
Alcohol Alcohol Respired
soluble insoluble s
Total 5 Standard
deviation
80.2 17.5 2 3 100.0
of individual
countings. 221
zt 52,220
87.1 11.6 1.3 loo.0 8R.T 14.3 2.0 1000 81.3 16.3 2.0 100.0
149,176 8,717 15,622
zk 10,254 f 2,921 X.86 f
173,515
+
10,688
100.0
506,314 10,971 29,711
f 21,12S f 1,376 zt 1,116
92.5 2.1 5.4
546,996
+
24,234
100.0
1 546,032 29,594 1 49,951 I ~~~ 1 625,577
f * *
23,295 1,476 2,084
87.0 5.0 8.0
+
24,554
100.0
’ old
loo. 0
3
soluble insoluhie
Total
16 days
90.5 7.5 2.0
P
7’flt:ll 16 da_\-s old
6,095Q 1,493 116
86 5 !I
-
222
NELSON, KROTKOV AND REED 60
.
20
200
__.-. 0‘
-------
-mm__
^.w_ 0
4
0
6
I2
b I
600
ti60
400
40
200
20 -.B -o-
wate -0
glucose 0 s
4 Time
I2
in hours
FIG. 2. A comparison water WOP
or radioactive derived from
of respiration of lupine seedlings infiltrated with either glucose. The dotted line indicates specific activity of the radioactive glucose.
RESULTS AND DISCUSSION
Respiration data were essentially the same in all of the experiments, and Fig. 1 shows a typical set of them. A summary of the distribution of radioactivity at the end of each experiment is given in Table II.
MET.4BOLISM
OF
RADIO.4CTIVE
223
ASPARAGINE
From Fig. 1, one can see that infiltration with asparagine versus water had little effect on CO2 production. From these data alone one might conclude that asparagine was not used as a respiratory substrate. The fallacy of this conclusion is revealed by the fact that the CO? respired was radioartive, even within the first 4 hr. after infiltration. As is seen from Table II, incorporation of asparagine into alcoholinsoluble materials was observed in every case. This was true even in TABLE Activity
in
Various
After
Fraclions
Lupine
III Znfiltration
seedlings.
with
Expt.
Fraction
Alcohol soluble Alcohol insoluble Respired Total
11 tlnys old
Alcohol soluble Alcohol insoluble Respired
Glucose
.4s percentage
Activity I~~-
4 days old
Radioactive
5
counls/min./jmclio?3
26,560 f 16,542 f 8,474 zt
2,213” 9 290
~
51,576 f
2,232
100
~
79,508 f 33,944 f 27,810 f
9,178 7,581 988
56 24 20
141,262 f
11,942
~ I
52 32 16
---
_~~ 37 days old
o Standard
~~~~
Total
’ Alcohol soluble Alcohol insoluble I Respired I Total deviation
of individual
I-~~-
66,028 f 34,256 f 26,183 f
798 7,699 503
100 ~
52 27 21
-__ 126,467 f 7,756 __ -.~~ .~
1OU
countings.
d-day-old lupine seedlings, which according to the generally held opinion, produce, but do not utilize asparagine. The amounts of asparagine used both in respiration and incorporated into alcohol-insoluble cell materials, were fairly uniform, varying in different experiments between 10 and 20%. In Espts. 1, 2, and 3, more asparagine was incorporated into cells than was respired, while the reverse was true in Expt. 4. This difference bet,ween the two sets of experiments might be due to the use of a different source of vermiculite to grow seedlings in Expt. 4. It has been found during this investigation that vermiculite has a considerable effect on
224
NELSON, KROTKOV .4ND REED
the growth. Thus, seedlings of comparable chronological age, grown on vermiculite from the second source, were about 25% taller. To evaluate asparagine as a respiratory substrate, it was decided to compare it with glucose. For this purpose lupine seedlings, grown in vermiculite from the same source as in Expt. 3, were infiltrated with 0.05 M radioactive glucose uniformly labeled (8). This glucose was checked chromatographically and found to contain about 15 % of radioactive fructose. The results of this experiment are given in Fig. 2 and Table III. Figure 2 reveals that unlike asparagine, glucose stimulates COZ production. Moreover, at the end of 12 hr., only half of the infiltrated glucose remained in the alcoholic extract, while the rest was either respired or incorporated into cells. A similar value for asparagine is SO-90 %. Lupine seedlings, therefore, utilize glucose 2x-5 times more rapidly than asparagine. But although glucose is used equally well either as a respiratory substrate or for the synthesis of cells, asparagine is usually used mainly for the production of the new cells. This conclusion is valid on the assumption that alcoholic extracts contained only infiltrated asparagine or glucose, and no other alcohol-soluble products into which these two compounds were changed by the cells. SUMMARY
1. Radioactive asparagine has been isolated from lupine seedlings, which were permitted to carry on photosynthesis for 6 days in the presence of CY402. It was found to contain 55% of its activity in the a-carboxyl. 2. Radioactive asparagine was vacuum infiltrated into wheat and lupine seedlings, and its subsequent metabolism was observed. 3. Utilization of asparagine for both CO? production and the synthesis of cells was observed in all cases. This was true even in young lupine seedlings before they develop their photosynthetic mechanism. 4. Radioactive glucose, infiltrated into lupine seedlings, was utilized 29&-5 times faster than asparagine. Although glucose was used about equally for respiration and cell synthesis, asparagine was used mainly for the synthesis of cells. REFERENCES 1. MEISS, A. N., C’onn. dgr. Expt. Sta. Bull. 663 (1952). 2. DUFF, G. H., AND FORWARD, DOROTHY F., Can. J. Res. C27, 125 (1949).
METr\BOIdSM
OF
R.\DIO.\(‘TIVE
‘,‘, i-., r
4SI’.\I<.lGINE
3. Wrcsox, D. (+., I\J. 4. Thesis, Queen’s I’niversity, Ontario, Canada, l!GO. 11. Cr., KROTSOV, G., AND REED, G. R., Science 113. 695 jl%il~.
4. WILSON, 5.
AhORE,
s.,
.4ND
6. I
~~ITTORIO,
1’.
STEIN, Science
INI) v..
74. 77.5 (19503.
w.
H.,
J.
Bid.
C’hl.
176,
367
(1(348).
106. 318 (19473.
SHIT, I’., ~
Can. G.,
29, 533 il951). REED. G. PI.. f’rw. sot.
J. (Xem. ASI)
ihpfl.
Rid.
Iled.
and Bacteriology,
Queen’s liniversily,
Kingston,
Ontario, Canada Received December 3, 1952 INTRODUCTION
The wide distribution and high concentration of asparagine in higher plants provide ample evidence of its physiological importance. Unfortunately, our present picture of its role has been derived mainly from the results of nitrogen analysis; and experimental data pertaining to the metabolism of its carbon chain, with a few notable exceptions (I), are still wanting. It has been repeatedly observed that, in the early stages of seed germination, asparagine accumulates irrespective whether seedlings are in light or in darkness. Its disappearance in later stages occurs only in light. From this observation it has been concluded that young seedlings can produce, but not utilize, asparagine, and that utilization of asparagine can take place only in light but not in darkness. It has been felt by the present authors that metabolism of asparagine carbon chain could be conveniently studied if CY4-labeledasparagine were introduced into plants. Moreover, with the help of such an asparagine one can also examine the effect of age and light on the utilization of this compound in plants. The experiments described below were performed with these objects in mind. MATERIALS Wheat leaves (Cornell 595) and blue lupine seedlings (Lupinus angustifolius) were used as experimental material. Both were grown in flats, containing loamy soil in the case of wheat, and vermiculite in the case of lupine. Flats, kept in a special room at 22 f 5”C., were under fluorescent lights for 16 hr. daily. First leaves of wheat were used when they were 14 days old. At this age they 1 Aided by a grant from the National Research Council of Canada. 2 Holder of an Ontario Research Council scholarship. 218
METABOLIS,M
OF RADIO.4CTIVE
219
.4SP,4RAGINE
hat1 reached their full size, and carried on photosynthesis (2). Lupine seedlings Tvere used at diflerent ages. It was shown previously that in blue lupine the photosynt.hetic meirhanism is already developed in 8-dav-old seedings (3). Preparation
of Radioactive
Asparaginc
Radioactive asparagine was produced by the lupiue seedings, which were placed in a desiccat,or with about 3 millicuries (mc.) of C’WQ and illuminated 16 hr. daily for 6 days (4). Following its isolation from the seedlings, asgaragine was recrystalieed aud its purity was checked chromatographically. Of its tot.:11activity, 1.6% was found to be in aspart,ic acid. A sample of this asparagine was dissolved in water, and three aliquots of it, w(Jrc degraded with uinhydrin to liberate a-carboxyl as carbon dioxide. Degradation was carried out under the conditions described by Moore and Stein (5). From each aliquot three plates were prepared and counted with a thin-window Geigerhliillrr t,uhe. The results are given in Table I. TABLE Radioactivity
of Asparagine
I
After Reaction with Xinhydrin of Moore and Stein (6)
Sk%tlple
Activity counlsjmin.
Before degradation After degradation Aliquot 1 Aliquot 2 Aliquot 3
718 ZlZ 4” 292 f 333 f 346 f
a Standard deviation of individual
6 15 16
IJnder the Conditions Per cent of activity lost
0 59 54 52
countings.
It is clear from these data that asparagine fined to the a-carboxyl. This lack of uniform whole chain of asparagine is surprising, since C1W2 xft.er 6 days of photosynthesis. Under euamplc, is lab&d uniformiv within 1 hr.
had about 55y0 of its activity COIIdistribution of Cl1 throughout the t,his ssparagine was produced from comparable conditions glucose, for
EXPERIMENTAL
A 0.05 111solution of radioactive asparagine was vacuum infiltrated into wheat leaves and lupine seedlings (6). These were placed in dark respiration chambers, kept at 20 f 2”C., and a stream of COz-free air was drawn over them. CO2 respired was collected in 0.05 N NaOH and determined by titration. Its specific activity wm determined following plating as BaC03. After three 4-hr. periods of respiration, or a total of 12 hr., plants were ext,rarted under a reflux condenser for 12 hr. with boiling 85 % ethanol and the extracts were made to volume. Three aliquots of each alcoholic
220
NELSON,
KROTKOV
AND
REED
I 8
4
100
IO
-O------O-specific 0
I 0
activity I
I 4
Tie
in hours
88
0
I2 I2
FIQ. 1. A comparison of respiration of lupine seedlings infiltrated with water or radioactive asparagine. The dotted line indicates specific activity CL402 derived from radioactive asparagine.
either of the
extract and alcohol-insoluble cell residue were completely oxidized by a wet-combustion method (7), and the CO%produced from each aliquot was plated in triplicate and counted as BaCOo.
_
~~-__--~
~... courr(s,mirr.,fracti~,~
ll~hed
14 &!JS soluble insoluble
113lliBh.,
Old.
E.,-jY,t.
f
71,620 .5,95s 1,52J1
f f f
79,099
z?z 6,277
Ih3~~irocl
io.094 15.322 2,009
* * f
3,048 2.238 111
Tot:tl
87,425
of;
3,iHX
,2looht,I Alcohol Itt*nIGrrcl
‘rc,t :11 r,
r,,rpin.c
seedlings.
s da\ s old
Expt.
Alcohol Alcohol Respired
Ercpt.
Alcohol Alcohol Respired
649 , 9’)O .k i6,630 . 86,810 zt 32,184 0;li4+ 240
-,
soluble insoluble
~~~... __.. ~~~~~~ :SO da?.8 old Alcobol soluble Alcohol insoluble Respired Tot al
1 746.274 I j 568,887 1 96,508 1 13,921
f
83,115
f f It
76,342 21,154 365
i
679,316
zk 711,220
324,086 66,058 8,6X!
f 42,979 zk 29,659 f z20
1 398,796
~,c~>ino seedZings. Ezpt. 4 4 da\-Y oh? Alcohol soluble Alcohol insoluble Respired ToM Alcohol Alcohol Respired
soluble insoluble
Total 32 days
old
Alcohol Alcohol Respired
soluble insoluble s
Total 5 Standard
deviation
80.2 17.5 2 3 100.0
of individual
countings. 221
zt 52,220
87.1 11.6 1.3 loo.0 8R.T 14.3 2.0 1000 81.3 16.3 2.0 100.0
149,176 8,717 15,622
zk 10,254 f 2,921 X.86 f
173,515
+
10,688
100.0
506,314 10,971 29,711
f 21,12S f 1,376 zt 1,116
92.5 2.1 5.4
546,996
+
24,234
100.0
1 546,032 29,594 1 49,951 I ~~~ 1 625,577
f * *
23,295 1,476 2,084
87.0 5.0 8.0
+
24,554
100.0
’ old
loo. 0
3
soluble insoluhie
Total
16 days
90.5 7.5 2.0
P
7’flt:ll 16 da_\-s old
6,095Q 1,493 116
86 5 !I
-
222
NELSON, KROTKOV AND REED 60
.
20
200
__.-. 0‘
-------
-mm__
^.w_ 0
4
0
6
I2
b I
600
ti60
400
40
200
20 -.B -o-
wate -0
glucose 0 s
4 Time
I2
in hours
FIG. 2. A comparison water WOP
or radioactive derived from
of respiration of lupine seedlings infiltrated with either glucose. The dotted line indicates specific activity of the radioactive glucose.
RESULTS AND DISCUSSION
Respiration data were essentially the same in all of the experiments, and Fig. 1 shows a typical set of them. A summary of the distribution of radioactivity at the end of each experiment is given in Table II.
MET.4BOLISM
OF
RADIO.4CTIVE
223
ASPARAGINE
From Fig. 1, one can see that infiltration with asparagine versus water had little effect on CO2 production. From these data alone one might conclude that asparagine was not used as a respiratory substrate. The fallacy of this conclusion is revealed by the fact that the CO? respired was radioartive, even within the first 4 hr. after infiltration. As is seen from Table II, incorporation of asparagine into alcoholinsoluble materials was observed in every case. This was true even in TABLE Activity
in
Various
After
Fraclions
Lupine
III Znfiltration
seedlings.
with
Expt.
Fraction
Alcohol soluble Alcohol insoluble Respired Total
11 tlnys old
Alcohol soluble Alcohol insoluble Respired
Glucose
.4s percentage
Activity I~~-
4 days old
Radioactive
5
counls/min./jmclio?3
26,560 f 16,542 f 8,474 zt
2,213” 9 290
~
51,576 f
2,232
100
~
79,508 f 33,944 f 27,810 f
9,178 7,581 988
56 24 20
141,262 f
11,942
~ I
52 32 16
---
_~~ 37 days old
o Standard
~~~~
Total
’ Alcohol soluble Alcohol insoluble I Respired I Total deviation
of individual
I-~~-
66,028 f 34,256 f 26,183 f
798 7,699 503
100 ~
52 27 21
-__ 126,467 f 7,756 __ -.~~ .~
1OU
countings.
d-day-old lupine seedlings, which according to the generally held opinion, produce, but do not utilize asparagine. The amounts of asparagine used both in respiration and incorporated into alcohol-insoluble cell materials, were fairly uniform, varying in different experiments between 10 and 20%. In Espts. 1, 2, and 3, more asparagine was incorporated into cells than was respired, while the reverse was true in Expt. 4. This difference bet,ween the two sets of experiments might be due to the use of a different source of vermiculite to grow seedlings in Expt. 4. It has been found during this investigation that vermiculite has a considerable effect on
224
NELSON, KROTKOV .4ND REED
the growth. Thus, seedlings of comparable chronological age, grown on vermiculite from the second source, were about 25% taller. To evaluate asparagine as a respiratory substrate, it was decided to compare it with glucose. For this purpose lupine seedlings, grown in vermiculite from the same source as in Expt. 3, were infiltrated with 0.05 M radioactive glucose uniformly labeled (8). This glucose was checked chromatographically and found to contain about 15 % of radioactive fructose. The results of this experiment are given in Fig. 2 and Table III. Figure 2 reveals that unlike asparagine, glucose stimulates COZ production. Moreover, at the end of 12 hr., only half of the infiltrated glucose remained in the alcoholic extract, while the rest was either respired or incorporated into cells. A similar value for asparagine is SO-90 %. Lupine seedlings, therefore, utilize glucose 2x-5 times more rapidly than asparagine. But although glucose is used equally well either as a respiratory substrate or for the synthesis of cells, asparagine is usually used mainly for the production of the new cells. This conclusion is valid on the assumption that alcoholic extracts contained only infiltrated asparagine or glucose, and no other alcohol-soluble products into which these two compounds were changed by the cells. SUMMARY
1. Radioactive asparagine has been isolated from lupine seedlings, which were permitted to carry on photosynthesis for 6 days in the presence of CY402. It was found to contain 55% of its activity in the a-carboxyl. 2. Radioactive asparagine was vacuum infiltrated into wheat and lupine seedlings, and its subsequent metabolism was observed. 3. Utilization of asparagine for both CO? production and the synthesis of cells was observed in all cases. This was true even in young lupine seedlings before they develop their photosynthetic mechanism. 4. Radioactive glucose, infiltrated into lupine seedlings, was utilized 29&-5 times faster than asparagine. Although glucose was used about equally for respiration and cell synthesis, asparagine was used mainly for the synthesis of cells. REFERENCES 1. MEISS, A. N., C’onn. dgr. Expt. Sta. Bull. 663 (1952). 2. DUFF, G. H., AND FORWARD, DOROTHY F., Can. J. Res. C27, 125 (1949).
METr\BOIdSM
OF
R.\DIO.\(‘TIVE
‘,‘, i-., r
4SI’.\I<.lGINE
3. Wrcsox, D. (+., I\J. 4. Thesis, Queen’s I’niversity, Ontario, Canada, l!GO. 11. Cr., KROTSOV, G., AND REED, G. R., Science 113. 695 jl%il~.
4. WILSON, 5.
AhORE,
s.,
.4ND
6. I
~~ITTORIO,
1’.
STEIN, Science
INI) v..
74. 77.5 (19503.
w.
H.,
J.
Bid.
C’hl.
176,
367
(1(348).
106. 318 (19473.
SHIT, I’., ~
Can. G.,
29, 533 il951). REED. G. PI.. f’rw. sot.
J. (Xem. ASI)
ihpfl.
Rid.
Iled.