Download 14C2H4 : Distribution of 14G-labeled tissue metabolites

Plant & CtUPkyiiol. 18: 141-148 (1977)
14
C2H4 : Distribution of 14G-labeled tissue
metabolites in pea seedlings l
Robert Giaquinta and Elmo Beyer, Jr.
(Received June 28, 1976)
The "C-metabolite distribution pattern following "CsH^ metabolism in intact pea
seedlings (/"warn satinum L.) was determined under various conditions. After a 24 hr
exposure to 14 CaH|, the majority of 14C-metabolitcs were water-ioluble (60-70%) with
lesser amounts in the protein (10-15%), lipid (1%), and insoluble (1-2%) fractions.
Ion exchange chromatography of the water-soluble components into basic, neutral, and
acidic fractions revealed a 50 : 40 : 10 distribution, respectively. Chromatography of
the neutral fraction revealed two regions of radioactivity (Rf-=0.38) and 0.63 which did
not cochromatograph with twenty-two known sugars or neutral metabolites. Chromatogramj of the basic fraction contained 3 regions of radioactivity. Similar distribution
patterns were noted when 14CjH< exposure was followed by a 6 hr air chase or when 5%
COi, an antagonist of cthylene action, was present during die exposure.
Marked differences in the 14C-metabolite distribution patterns were obtained when
14
COa was substituted for 14 CjH4. These results indicate that the metabolic pathway
involved in ethylcne metabolism is different from that involved in intermediary carbon
metabolism.
The early literature on ethylene metabolism is marked with controversy as
to whether or not radiolabeled ethylene is incorporated and metabolized by plant
tissues (6-11, 13, 14). Much of this uncertainty concerning the metabolic fate of
ethylene possibly existed because stringent precautions were not taken to eliminate
radioactive impurities from the labeled ethylene and to minimize microbial utilization of the labeled gas (1, 3-5). Because of these criticisms the validity of these
earlier studies has been questioned (1).
Recently, a gas chromatographic purification technique was described for
obtaining ultra high-purity "CgHU (3). Plant metabolism studies (4, 5) with this
purified material have now conclusively demonstrated that intact aseptically grown
pea seedlings incorporate and metabolize 14C2H4 into tissue components and
14
CO a .
In the present study, using high purity ethylene and aseptic techniques, ion
exchange fractionation and chromatography techniques were used to examine the
distribution pattern of 14C metabolites derived from HC2H4 incorporation into
1
Contribution No. 2338 from Central Research and Development Department, Experimental Station,
E. I. du Pont de Nemours and Company, Wilmington, Delaware.
141
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Central Research and Development Department, Experimental Station
E. I. du Pont de Nemoun and Company Wilmington, Delaware 19898, U. S. A.
142
R. Giaquinta and E. Beyer, Jr.
pea seedlings. The results indicate that the metabolic pathway for ethylene incorporation and metabolism is unique and not common with normal intermediary
carbon metabolism.
Materials and methods
u
CzHi purification and treatment
14
Analytical methods. After exposure of pea seedlings to cither 14C2H4 or 14 COj,
9 seedlings (ca. 5 g fr.wt.) were rapidly frozen in solid COa. The tissue was later
homogenized in a mortar and pestle with hot, 80% ethanol (v/v), and extracted
three times with 200 ml aliquots of boiling 80% ethanol. Centrifugation at 15,000 X
g for 30 min yielded the alcohol-soluble fraction and the alcohol-insoluble pellet
containing starch, precipitated proteins, and other insolubles.
The alcohol-soluble fraction was extracted with two volumes of petroleum
ether to yield the lipid fraction. The water-soluble material was further fractionated by sequential ion exchange chromatography using 1x8 cm columns of
AG50W X 8 (H+; 200-400 mesh) and AG-1-X8 (formate; 200-400 mesh) as
cation and anion exchange resins, respectively (2). Four fractions were obtained
from the coupled column: (1) the neutral fraction (mainly sugars) eluted with
60 ml of H2O through both columns; (2) the basic fraction (mainly amino acids)
eluted from the cation column with 50 ml of 2 N NH4OH or 2N HC1; (3) the acid-I
fraction (organic acids, sugar monophosphates and other weakly acidic compounds)
eluted from anion column with 50 ml of 6 N formic acid and (4) the acid-II fraction
(strongly acidic compounds, i.e., phosphoglyceric acid and sugar diphosphates)
eluted from the anion column with 50 ml of 2 N HC1.
The above eluants were evaporated to dryness in vacuo at 35°C, redissolved
in 1 ml of water, and aliquots scintillation-counted to determine the distribution
of 14C-labeled products. Recovery from the columns was greater than 90% as
determined with 14C-standards. Sugars in the neutral fraction were further
resolved by one-dimensional descending paper chromatography on 3 MM Whatman
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CaH4 purification, aseptic culture of pea seedlings (Pisum sativum, cv. Alaska)
and their treatment with 14C2H4 under aseptic conditions were according to
procedures described recently (3-5). Briefly, uniformly labeled ethylene was purified
by gas chromatography by sequential passage through a silver nitratc-ethylene
glycol coated Gas Chrom R and Porapak T columns. 14CgH4 was collected and
stored over 0.5 N NaOH to remove 14G radiolysis products actively incorporated
by the seedlings. Three two-day old etiolated pea seedlings were incubated in
closed flasks containing 3 ml of HSO with 80 to 100/il/liter of "C2H4 (ca. 12 X
106dpm) for 24 hr at 27°C in the dark. The actual CaHi concentration was
determined by gas chromatography at the end of the 24 hr exposure. As previously
described (5), a NaOH trap was present within the incubation flask to collect the
14
CO2 released from tissue-dependent ethylene oxidation. In some experiments
5% COa (v/v), an antagonist of ethylene action, was present during the l4C2H4
exposure (5). To distinguish the incorporation of 14C2ti4 from COa metabolism,
experiments were also conducted in which pea seedlings were exposed to 14CO2
(initial concentration of 300 /xl/liter, 6.5xl0 6 dpm) with or without 80/il/liter
of nonlabeled C2H4 for 24 hr.
i«CiH4 metaboliim
143
Results and discussion
Previous studies (4, 5) have shown that etiolated pea seedlings actively
metabolize 14C-ethylene. This metabolism consists of two components: an oxidation of14C2H4 to 14COa with a concomitant incorporation of14C into unidentified
tissue components. The distribution pattern of 14C-labeled tissue metabolites
obtained by solvent extraction and ion exchange chromatography after a 24 hr
exposure to 14C2Hi under various conditions is shown in Table 1. The majority
of the radioactivity from 14C2H4 (60 to 70%) was in the water-soluble metabolites
with lesser amounts in the protein (10 to 15%), lipid (<1%), and insoluble (1 to
2%) fractions (Table 1, column 1). Further separation of the water-soluble fraction
by ion exchange chromatography revealed the following distribution: basic fraction,
mainly amino acids, 50-55%; acid fraction 8 to 10%, the majority present in the
acid-I eluant comprised of organic acids, sugar monophosphates, and other weakly
acidic components; and the neutral fraction, mainly sugars, 35 to 40%. Table 1
also shows the specific activity of the tissue incorporation in relation to that of the
14
CO2 released during ethylene oxidation. The levels and distribution of specific
activities agree well with those reported previously (5).
The pattern of 14C-metabolite distribution and the level of tissue incorporation
do not change following a 6 hr exposure to air after the 14C2Hi treatment (Table 1,
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paper (20x50 cm) for 15hr using /z-butyl alcohol: acetic acid : H 8 O ( 3 : 3 : 2 ,
v/v). The neutral fraction was also separated by TLC using pre-coated cellulose
300 MN plates in an ethyl acetate : acetic acid : formic acid : water ( 1 8 : 3 : 1 : 4 ,
v/v) solvent system. Sugars were detected with Sigma aniline diphenylamine spray
and identified by cochromatography with appropriate standards.
Amino acids in the basic fraction were separated by one-dimensional descending
paper chromatography (Whatman 3 MM paper, 20 X 50 cm) using three irrigations.
The- solvent system was n-butyl alcohol : acetic acid : H2O ( 9 : 1 : 2.5, v/v) and
amino acids were detected with ninhydrin reagent and identified with known
standards.
Profiles of radioactivity of the neutral and basic chromatograms were determined by dividing the unsprayed chromatograms into 1 cm sections from the
origin to front, and extracting the segments in liquid scintillation vials containing
1 ml of H2O. Scintillation fluor was added and the radioactivity determined.
14
C-standards revealed no loss in counting efficiency and recovery was 100% by
the above procedure.
The alcohol-insoluble pellet obtained from the initial tissue extract was fractionated into proteins and starch components (12). The protein fraction was
obtained by extracting the pellet four times with 30 ml aliquots of 0.1 N NaOH
(approx. 20 min each). The protein was then precipitated with 10% (v/v) trichloroacetic acid from the NaOH supernatants, centrifuged at 20,000 X^, and washed
once with 5% (v/v) TCA. The protein was dissolved in buffer and its radioactivity
determined. The insoluble pellet (starch and other insolubles) was dried at 90°C
overnight and combusted to 14COg in an Intertechnique oxymat; the 14CC>2 was
collected in a phenethylamine-toluene cocktail and counted via liquid scintillation
spectroscopy.
R. Giaquinta and E. Beyer, Jr.
144
Table 1 Metaboliit distribution of 1*C-/aW«/ products qfitr 1*C-t0tfUm or uCOt exposure to pea seedlings
for 24 hr
Intact
"CH4
1.5
12
<1.0
62
Basic
56
8
Acid*
Neutral
36
Unidentified
4
% Recovery
80
Specific activity of
" C in total tissue'
141
/dpm/mg dry\
\wt./24 hr
)
Specific activity of
" C in NaOH
/dpm/mg dry\
iwt/24 hr
j
*
*
'
*
284
1.0
13
<1.0
62
57
8
35
1.2
11
<1.0
70
52
7
41
8
7
84
91
138
126
19.4e
• Homogenized
"C1H4
"COa
+5%COi
- l s CsH4
% Distribution
18.5
2
15
<1.0
"COt
"C2H4
75
<1.0
74
2
34
<1.0
45
70
69
45
29
1.0
2
95
30
1.3
10
45
1685
-<
2
10
3
3
90
85
1905
32
4
1.3
Acdd-I fraction contained 90% of the "C-labcl (See Materials and methods).
Dry weight of total tissue (9 seedlings) was 1260 mg.
During 6 hr air chase.
NaOH omitted from flasks.
column 2) suggesting that these metabolites represent terminal pools or pools with
a slow turnover rate. The specific activity of the 14 COa trapped into NaOH
represents the amount of 14COa released during the 6 hr air chase (minus "CgH*).
Most of this probably represents a physical diffusion of 14 COa from the tissue and/or
water surrounding the tissue.
Carbon dioxide, a known antagonist of ethylene action, was previously shown
to markedly inhibit the oxidation of 14CgH4 to 14 COa without affecting 14C-tissue
incorporation (5). Similar results are noted in Table 1, column 3, in which 5%
COa inhibited 14 COa release greater than 90% without significantly altering
14
G-tdssue incorporation. In addition, 5% COa had no influence on the distribution
of 14 C-label derived from 14CaH4 into various metabolites. This different sensitivity
of CaH4 oxidation and tissue incorporation to 5% COa indicates that tissue incorporation is not dependent on 14CaH4 oxidation to 14 COa. Furthermore, since
14
C2H4 incorporation occurred without 14CaH4 oxidation to 14 CO2, the tissue 14 C
could not be the result of refixation of 14 COa released during "CatLj oxidation.
Further evidence against the refixation of 14 COa by tissue carboxylating reactions
is presented in the following experiments using 14 COa in place of 14CaH4.
Pea seedlings were exposed to 300 /*l/liter 1 4 C O E , in the presence and absence
of 80 ul/liter nonlabeled ethylene to determine whether the incorporation pattern
of 14G was similar to that observed for 14 C 2 H4 incorporation. Table 1, columns
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Insoluble (starch)
Protein
Lipid
Water soluble
then Air
(6hr)
metabolism
145
S
i
300
10
Q F
t I
X
Y
1
15
20
23
30
DISTANCE FROM ORIGIN (cm)
NEUTRAL
FRACTION (a)
I4
CtH4
33
40
431
FRONT
Fig. 1. Distribution of radioactivity after chromategraphjr of the ntutral fraction following a 24 hi exposure to (a)
u
CtHt and (A) 1*C0%+laCtHi. S, G, F, are sucrose, glucose and fructose, respectively. X and Y are
unknown.
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4 and 5, show that marked differences existed in the metabolite fractions and in
the amount of14C incorporated into the total tissue. When 14COa was the substrate
(with or without 14C2H4), 30% of the 14C in the water-soluble fraction was in
acidic compounds while the neutral fraction comprised only 1% of the label. In
contrast, the reverse was found for 14C2H4 incorporation (Table 1, columns 1-3).
That is, the acid fraction contained 5 to 10% of the label while the neutral fraction
represented 35-40% of the 14C water-soluble materials. No differences in 14C
distribution were noted for 14COz with or without nonlabeled ethylene indicating
that the differences observed between 14CsH4 and 14CC>2 incorporation could not
be attributed to an ethylene-induced alteration of14CC>2 metabolism. Major differences were also noted for the amount of 14G incorporated by the tissue after 14CC>2
and 14CaH4 exposure. Tissue exposed to 14CaH« incorporated ten-fold less 14C
than those exposed to 14CC>2 even though the initial specific activity of the 14C2H4
was 6-fold greater than that of the 14CO2- The differences in 14C metabolite
distribution and levels of 14C incorporation following 14CC>2 and 14C2H4 exposure
indicate that the pathway(s) involved in 14C2H4 metabolism differs from that
involved in intermediary carbon metabolism.
Previously it was shown that homogenizing the tissue in 1 ml of HgO prior to
14
C2H4 exposure completely prevented 14C2H4 oxidation to 14COg and inhibited
tissue incorporation by approximately 60% (5). A similar response is shown in
Table 1, column 6, in which homogenization inhibited tissue incorporation by
approximately 75% while the inhibition of 14CO2 evolution was virtually complete.
The major difference in the metabolite distribution pattern of 14C under these
146
R. Giaquinta and E. Beyer, Jr.
sucrose
lactose
maltose
stachyose
rafRnose
glucose
fructose
galactose
mannose
sorbose
arabinose
xylose
fucose
ribose
deoxyribose
erythrose
rhamnose
mannoheptulose
seduheptulose
glyceraldchyde
dihydroxyacetone
glycerol
The chemical presence of components X and Y was indicated by aniline
diphenylamine with or without ethylene treatment and thus their presence is not
dependent upon the addition of exogenous ethylene.
The two unknowns, X and Y, were not visualized by />-anisidine-HCl, another
sugar reactive agent, even though the radioactivity profile revealed their presence.
Neither did these labeled components react with indicators for sugar alcohols
(potassium permanganate), amino sugars (ninhydrin), nor reducing sugars (aniline
hydrogen phthalate) (15). A second passage of neutral fraction through the coupled
cation and anion exchange columns did not alter the radio- or chemical-presence
of X and Y indicating they are neutral compounds, not necessarily sugars, and not
anionic or cationic contaminants. The two unknowns also could not be identified
by gas chromatography or field desorption and chemical ionization mass spectroscopy.
Fig. la, b shows the marked differences between the radioactivity profiles
of the neutral fractions of the "CO2 (with "C2H4) and 14C2H4 treated tissue.
Unlike 14C2H4 exposure (Fig. la), sucrose is the major labeled species of the neutral
fraction when 12COa is applied with some radioactivity in component X. A similar
profile was obtained in the absence of nonlabeled C2H4 (not shown) indicating
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conditions was a 2- to 3-fold increase in the amount of 14C present in trichloroacetic
acid-precipitable proteins. There was also an equalization in the percent of
radioactivity appearing in the basic and neutral fractions. The significance of
this is unknown.
Since the majority of the 14C-label in the water-soluble fraction (90%) was
in neutral and basic compounds, these fractions were further separated by paper
chromatography. Fig. 1 (a, b) shows the radioactivity profile obtained from a
chromatogram of the neutral fraction following tissue exposure to 14C2H4 and
14
COa (+14C2H4) for 24 hr. In both exposure treatments, there were five components visualized after spraying with the carbohydrate detecting aniline diphenylamine. Aniline diphenylamine forms various color reaction products with different
sugars. The three major components were readily identified as sucrose (being the
most predominant), glucose and fructose with Rf values of 0.22, 0.28 and 0.32,
respectively. The radioactivity profile (Fig. la) shows two regions of activity which
corresponded with two components, X and Y, having Rf values of 0.38 and 0.63,
respectively. These radiochemical components gave a yellow color after reaction
with aniline diphenylamine and did not cochromatograph and/or give a similar
color product with any of the following sugars or neutral metabolites:
14
400
CaHi metabolism
JCYS
em
t
6LY
» HOM
147
BASIC FRACTION (a)
I4
CtH4
300
200
1500
1000
300
K)
13
20
25
30
33
DISTANCE FROM ORIGIN ( c m )
40
451
FRONT
Fig. 2. Distribution of radioactivity afttr chromalography qftfu basic fraction following a 24 hr txposure to (a)
14
Ct//« and (4) 1 < COi+ l a &i/.t. Cys, gin, gly and horn are cysteine, glutamine, glycine, and homoserine, respectively.
that G2H4 had no effect on the distribution of label within the neutral fraction.
These data demonstrate that 14CaH4 is metabolized by pea to two primary neutral
components with component Y being unique to "CgHj metabolism.
Table 1 showed that the basic or amino acid fraction contained 60 to 75%
of the 14C present in the water-soluble materials after both "CaH^ and 14CO2
incorporation. However, the distribution of label in the basic components differed
between the two exposures (Fig. 2 a, b). A 24 hr exposure to 14COa results in
homoserine being the predominant labeled amino acid followed by glutamine and
glycine (Fig. 2b).' Aspartate and serinc which migrate with glycine in this solvent
system, could also be present. Pea seedlings are unique in their amino acid metabolism in that homoserine is the major amino acid present during early developmental stages {12). Larson and Beevers (12) reported a similar 14C distribution
into amino acids following a 24 hr exposure of 2- to 3-day old pea seedlings to
14
C-glucose, suggesting this profile is a good representation of that accompanying
intermediary carbon metabolism. The distribution was the same for 14CC>2 minus
C2H4 exposures.
The distribution pattern of 14C into components of the basic fraction (not
necessarily amino acids) following 14C2H4 exposure differed markedly from that
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BASIC FRACTION (b)
l4
C0t + ItC2H4
148
R. Giaquinta and E. Beyer, J r .
The expert technical assistance of Adelita Burr and Richard Evans ii appreciated.
Mrs. Doris Bacon for the manuscript preparation.
We thank
References
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( 2) Atkins, C. A. and D. T. Canvin: Photosynthesis and COi evolution by leaf disci: ga» exchange,
extraction, and ion exchange fractionation of M C-labelcd photosynthctic products. Can. J.
Bot. 49: 1225-1234 (1971).
( 3) Beyer, E. M., Jr.: i«CaH«: Its purification for biological studies. Plant Physiol. 55: 845-848
(1975).
( 4) Beyer, E. M., Jr.: nC-ethylenc incorporation and metabolism in pea seedlings. Naturt 255:
144-147 (1975).
( 5 ) Beyer, E. M.F Jr.: ^CsH*: Its incorporation and metabolism by pea seedlings under aseptic
conditions. Plant Physiol. 56: 273-278 (1975).
( 6) Buhler, D. R., E. Hansen and C. H. Wang: Incorporation of ethylene into fruits. Nature 179:
48-49 (1957).
( 7 ) Carlton, B. C , C. E. Peterson and N. E. Tolbert: Effects of ethylene and oxygen on production
of a bitter compound by carrot roots. Plant Physiol. 36: 550-552 (1961).
( 8 ) Hall, W. C , C. S. Miller and F. A. Herrero: Studies with "C-labeled ethylene. In 4th Inttm.
Con/. Plant Growth Regulation. Iowa State University Press, Ames. pp. 751-778 (1961).
(9) Jansen, E. F.: Metabolism of labeled ethylene in the avocado: appearance of tritium in the
methyl group of toluene. J. Biol. Chan. 238: 1552-1555 (1963).
(10) Jansen, E. F.: Metabolism of labeled ethylene in the avocado. II. Benzene and toluene from
cthylene-"C; benzene from ethylene-3H. J. Biol. Ounu 239: 1664-1667 (1964).
(11) Jansen, E. F.: The metabolism of ethylene in the avocado. In Food Scitnct Technology, Vol. 1.
Proc. 1st Intern. Congr. Food Sd. Tech., London. Edited by J. M. Leitch. Gordon and Breach,
Science Publishers, Inc. New York pp. 475-481. 1969.
(12) Larson, L. A. and H. Beevers: Amino acid metabolism in young pea seedlings. Plant Physiol.
40: 424-432 (1965).
(13) Shimokawa, K. and Z. Kasai: A possible incorporation of ethylene into RNA in Japanese
morning glory seedlings. Agr. Biol. Chem. 32: 680-682 (1968).
(14) Shimokawa, K., K. Yokoyama and Z. Kasai: Fixation of ethylene- u C by Japanese morning
glory seedlings (Pharbitis nil Chois). Mem. Pus. Inst. Food. Sd. Kyoto Univ. 30: 1-7 (1969).
(15) Stahl, E.: Thin-Loytr Chromaiography. Springer-Vcrlag, New York, 1969.
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obtained when 14COa was applied (Fig. 2a, b). The homoserine and glycineaspartate-serine regions contained little radioactivity. Instead, a broader region
of radioactivity is present in a component (s) migrating 4 to 5 cm ahead of homoserine in the region of glutamate or alanine. Another region of radioactivity which
differs from the 14COj profile, corresponds to the cystine-cysteine region. No
radioactivity was present in the region of methionine, a precursor of ethylene
biosynthesis, thus ruling out the possibility that 14C2H4 incorporation resulted from
the reversal of the ethylene biosynthetic pathway. Further identification of the
smaller regions of radioactivity could not be achieved because of the low levels of
radioactivity and high endogenous levels of amino acids present in the cotyledons.
While the exact chemical nature and physiological significance of the ethylene
metabolites in pea are unknown, the above data clearly indicate that the metabolic
pathway responsible for their formation is unique to ethylene metabolism. None
of the ethylene metabolites were found to possess significant growth regulating
activity when bioassayed on etiolated peas. These results, plus the slow turnover
rate observed during the 6 hr air chase (Table 1), suggest that these metabolites
are stable, terminal products of ethylene metabolism. Nevertheless, establishing
their chemical nature will undoubtedly shed light on the biochemical pathway
responsible for their formation. In turn, this may provide insight into the mechanisms of ethylene action if this metabolic system is linked to ethylene action as
previously suggested (4, 5).