Download 13-Alanine as an Ethylene Precursor

Planit Physiol. (1969) 14, 1217-1226
13-Alanine as an Ethylene Precursor. Investigations Towards Preparation,
and Properties, of a Soluble Enzyme System From a Subcellular
Particulate Fraction of Bean Cotyledons1
Robert A. Stinson' and Mary Spencer
Plant Biochemistry, South Laboratory, Universitv of Alberta, Edmonton, Alberta, Canada
Received Janluary 27, 1969.
Abstract. A method is described for the preparation, from a subcellular particulate fraction
of wax bean cotyledons, of a soluble enzyme system that is capable of converting 3-alanine
to ethylene. In the presence of ATP, CoA, thiamine pyrophosphate, MgSO4, and pyridoxal
phosphate, ethylene production is maximum at a 0.5 mNi concentration of 8-alanine. The
system exhibits a pH optimum at 7.0 but when the pH is raised above 8, evolution of the
volatile again increases and continues to do so up to pH 12. The enzyme system is stimulated
by either NADPH or NADH; the concentration of NADPH necessary to obtain maximum
activity is twice that of NADH. The requirement for a reducing agent is in agreement with
the proposal that malonate semialdehyde, formed by an aminotransferase reaction from
f3-alanine, is reduced to 83-hydroxypropionate. Both malonate semialdehyde and /-hydroxypropionate are better stimulators of production of the volatile in the soluble system than is
f3-alanine, and .8-hydroxypropionate is a better stimulator than malonate semialdehyde. This
system is also able to incorporate tritium from tritiated water into ethylene; this supports the
proposal that ethylene is formed by the decarboxylation of acrylate, the latter being formed
from f3-hydroxypropionate.
Experiments with both cold and labeled malonate suggest that this compound stimulates
ethylene production by acting as an end product inhibitor that prevents the loss of 'Malonate
semialdehyde from the pathway. Malonate does not appear to serve as a precursor.
Addition of cytopl-asmic enzymes to the 'soluble system' (prepared from particulate
enzymes) results in a considerable boost in ethylene production, but the specific activity
(mdul/mg protein) is lowered from that of the particulate enzymes alone.
Several compounds have been proposed as fairly
immediate ethylene precursors, including methionine,
methional, propanal (12), acetaldehyde (20, 21), and
,8-alanine. Although many workers have investigated the first 2 compounds a niumber of difficulties
remaini. MIethionine was found to stimulate ethylene
biosynthesis in aged apple tissue slices (14), in
indoleacetic acid treated pea stem segments. and in
apple and green banana sections (1). However,
Gaillard et al. (5) were unable to stimulate ethylene
production with methionine in either fresh or aged
discs of peel from pre-climacteric fruit even though
the discs, when aged, produced ethylene. The
stimulation that methional imparted to cell free
systems of cauliflower florets ( 15, 16, 17/) and pea
seedlings (14) led to the postulation of methional
as an intermediate between methionine and ethvlene.
However, methional, unlike methionine, would not
stimulate ethylene evolution from apple sections (13).
Thompson aind Spencer (26, 27) provided evidence to support a role for 8-alanine as an ethylene
precursor in hean cotyledons. They proposed that
the conversion of fl-alanine to ethylene involved the
intermediates malonate semialdehyde, 8-hydroxypropionate, and acrylate. Malonate was involved ir. a
side path throtugh malonate semialdehyde (Fig. 1).
+2H
Ii
semi -
propionate
III +2H+
aldehyde
-2H +||
Malonate
Iii
propionato
FIG. 1. Proposed pathway for conversion of
nine to ethylene.
8-ala-
This report describes experimiients that support
the pathway of Thompson and Spencer. The preparation of a soluble systenm, from a .subcellular
particulate fraction of wax bean cotyledons, capable
of deriving ethylene from ,l-alanine is detailed and
certain of its properties are described.
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The authors are grateful to the National Research
Council of Canada for a grant-in-aid (A-1451) of this
research and for a studentship to R.A.S.
2 Present address:
Department of Biochemistry, University of Bristol, Bristol, England.
1
-CO2
-H20
$-alanin= Malonate =-hydroxy- =Acrylate-e Ethylone
Copyright © 1969 American Society of Plant Biologists. All rights reserved.
1218
PLANT PHYSIOLOGY
Materials and Methods
Chemicals. Malonate-2-14C (>99 %) was obtained from New England Nuclear; ATP (A grade),
thiamine pyrophosphate (A grade), a-ketoglutarate
(A grade), and dithiothreitol from Calbiochem;
Triton X-100 from Applied Science Laboratories;
Sephadex G-25 from either Pharmacia or Sigma
Chemical Company; pyridoxal phosphate from Nutritional Biochemicals; TES was purchased from
General Biochemicals and recrystalized from ethanol;
tritiated water (99 %) from Volk Radiochemicals;
ethylene-'4C(U) (>98 %) from Nuclear Chicago
Corporation. NADPH and NADH were from
Sigma Chemical Company; silica gel G from
Canadian Laboratory Supplies; and sodium acrylate
from K and K Company. Other materials were
from Fisher Scientific Company Ltd.
Grouwth of Seedlings. Seeds of Phaseolus vulgaris L. var. Kinghorn wax were planted in horticultural grade vermiculite and grown at 26.00 in the
dark. The cotyledons were picked on specific days
after planting.
Preparation of Mitochondria and the Lyophilized
Subcellular Particulate Fractioni. Mitochondria were
isolated at 10,000g and washed as outlined by Stinson
and Spencer (22). The subcellular particulate fraction was isolated at 32,000g from wax bean cotvledons that were 3 and one-half days old, an age when
the cotyledons were producing significant amounts
of ethylene. The cotyledons were crushed by a meat
grinder in a buffer of the following composition:
0.3 M mannitol, 0.05 M TES [N-tris-(hydroxymethyl) methyl-2-aminoethanesulfonic acid] -NaOH
buffer, pH 7.4 at 00 l(2 ml buffer/g cotyledons).
The brei was filtered through cheesecloth and the
resulting filtrate subjected to differential centrifugation. The supernatant from the first spin at 2500g
for 10 min was centrifuged at 32,000g for 15 min.
The pellet obtained was resuspended in 0.01 M TES,
pH 7.6 at 0" (1 ml/8 g cotyledons) and freeze-dried.
This lyophilized fraction will be referred to as the
'crude enzyme'.
Solubilization of the Crude Enzyme. Two g of
lyophilized sub-cellular fraction (crude enzyme) were
solutbilized at 0° by treatment with 40 ml of 0.4 %
Triton X-100 (octylphenoxy,polyethoxy ethanol) in
0.01 M TES, pH 7.6 at 00 for 1 hr and then centrifuged. The supernatant layer from centrifugation
at 100,000g for 30 min was chromatographed on
Sephadex G-25 with 0.1 % Triton X-100, 0.01 M
TES, pH 7.6 at 00 solution as elution buffer. The
pellet was resuspended in 35 ml Triton buffer and
allowed to stir gently overnight at 00. Subsequent
centrifugation at 100,000g and gel filtration were as
above. The 2 filtrates comibined will be referred to
as the 'soluble enzyme system'.
Ethylene Analyses. Ethylene was collected by
passing purified air over the sample, through lithasorb to remove CO2, and through 'drierite', to remove
HoO. Ethylene was adsorbed on 0.5 g silica gel
(Davison, grade 15, 35-60 mesh) in a U shaped
glass tube (4 mm ID) in a dry ice acetone bath.
The tube was maintained at this temperature and
connected to a 2-way valve inserted in the helium
input line of a Perkin Elmer Model 811 flame
ionization gas chromatograph. With the valve
closed the tube was heated to 400 thereby releasing
the ethylene. The valve was subsequently opened
and the h-elium was rerouted through the silica gel
and onto a 37 cm X 6 mm (ID) column of activated
alumina coated with 2 and one-half percent Silicone
550. Column and detector temperatures were approximately 200 and 1250, respectively. He, H2,
and air flow rates were 64 ml/min, 37 ml/min, and
200 ml/min, respectively. For radioactive determinations, a flow splitter was utilized to direct 80 %
of the sample to a Nuclear Chicago gas flow counter
(gas radiochromatograph) and the remaining 20 %
to the flame of the gas chromatograph. The 85 ml
chamber of the gas radiochromatograph, when
flushed witlh high purity methane at 30 ml/min and
connected to a 10 mV potentiometric recorder, provided a continuous record of radioactivity in the
column effluent. The instrument was standardized
by injecting known amounts of ethylene-14C (U)
into the gas chromatograph and plotting peak height
vs. dpm of ethylene.
Preparatiwn of a Cytoplasmic Enzyme System.
The supernatant layer (cytoplasmic enzymes), after
isolation of the particulate fraction at 32,0O0g, was
brought to pH 7.6 at 00 and freeze-dried. Eighty ml
of buffer i(10 mm TES, 0.1 % Triton X-100, pH 7.6
at 00) were added and the insoluble material removed by centrifugation. The supernatant layer
from this centrifugation was chromatographed on
Sephadex G-25.
Hydrazone Formation and Thin-Layer Chroma-
tography. Four ml of reaction mixture that contained 1 ,uc malonate-2-14C, cofactors and enzyme
solution were removed after 2 hr of incubation and
the protein precipitated with trichloroacetic acid. A
small amount of cold malonate semialdehyde and
excess 2,4-dinitrophenyl hydrazine (recrystallized
from ethanol) solution [0.2 % (w/v) in 2 N HCl]
were added to the protein free sample. Extraction
with chloroform :ethanol (4:1) of the hydrazones
formed, evaporation to dryness under N2, and addition of 1.0 ml of solvent provided a sample suitable
for thin-layer chromatographic spotting on glass
plates coated with 0.5 mm silica gel.
Synthesis of Malonate Semialdehyde and ,B-hydroxypropionate. Both compounds were synthesized
according to the methods of Robinson and Coon
(19). Malonate semialdehyde was Fred in solution at -200 as sodium /3-hydroxypropionate.
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Copyright © 1969 American Society of Plant Biologists. All rights reserved.
STINSON AND SPENCER-f3-ALANINE AS AN ETHYLENE PRECURSOR
Results and Discussion
Respiration, With Malonate or Propionate as
Substrate, of Mitochondria From Cotyledons of
Germlinating Bean Seedlings. Since it has been
suggested that propionic acid may be involved with
ethylene biosynthesis (Introduction), the ability of
Anitochondria to metabolize this acid was investigated. Propionate has been shown previously (6, 8)
co be metabolized by mitochondria; present experiments with wax bean co.tyledon mitochondria were
designed to investigate the relationship between
propionate metabolism by mitochondria and ethylene
production by a subcellular particulate fraction that
was largely composed of mitochondria. Malonate,
which can be formed from malonate semialdehvde
(6), another compound on the proposed pathway
from f3-alanine to ethylene, was similarly studied.
Fig. 2 shows that as germination proceeds, the
ability of the mitochondria to metabolize the 2
substrates increases. It is perhaps significant that
ethylene evolved on a per g subcellular particulate
fraction protein basis also increases during germination (Stinson and Spencer, unpublished).
Thompson (26) reported that the levels of
malonate in the cotyledons of germinating beans
11
c
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10
0
C.
9
O PROPIONATE
* MALONATE
7
E
E
6
uJ
5
4
z
Lu
0
3
x
.0
2
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0
1
2
I
3
4
I
5
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I
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6
7
8
9
I
10
11
12
I
13
AGE (days)
FIG. 2. Respiration rates, with malonate or propionate as substrate, of mitochondria isolated from cotyledons of germinating bean seedlings. Assay medium:
17 mM malonate or 34 mm propionate, 0.3 M mannitol,
4 mm MgC12, 2.5 mm KH2PO4, and 1 mm TES, pH
72. Final volume was 3.2 ml and assay temperature
24 .
1219
increased almost linearly between the fourth and
ninth days after planting. It is quite plausible then
that both malonate levels and the ability of these
cotyledons to metabolize malonate could exert control over the biosynthesis of ethylene in this tissue.
Acetyl CoA is produced by plant tissue in the
metabolism of both propionate (8) and malonate
(7). The similarity in tihe respiration profiles of
these 2 substances certainly suggests that their
meta-bolism could occur through a common metabolite.
The metabolism of propionate through acrylate,
fl-hydroxypropionate, and malonate semialdehyde to
acetyl CoA constitutes a modified ,8-oxidation scheme
for the odd carbon numbered fatty acid, propionaite.
As mentioned, several of the enzymes in this conversion are also part of the proposed scheme for
conversion of fl-alanine to ethylene. Thus, the
ability of isolated mitochondria to metabolize propionate indicates that at least a portion of the scheme
for conversion of /8-alanine to ethylene 'is operative
in these cotyledons.
Substrate and Cofactor Stimulation of Ethylene
Production Fromt a Lyophilized Particulate Fraction.
Table I shows the stimulation in ethylene.production
obtained when the cofactors and substrate necessary
for the conversion of /8-alanine to ethylene (26)
were added to a suspension of 0.5 g lyophilized
particulate fraction (crude enzyme system). From
both experiments, it can be seen that the added
compounds caused a significant increase in ethylene
production especially on the 2 to 4 hr collection.
The increase in ethylene evolution brought about by
the addition of only /8-alanine ranged between 40 %
and 80 % of the total stimulation. The only other
compound found to stimulate evolution was malonate
bu.t variability in results did not allow a comparison
of the 2 compounds as precursors. Evidence is
given below, however, to show that malonate carbons
are not incorporated into ethylene. The larger
stimulation on the 2 to 4 hr collection suggests that
substrate(s) used up on the 0 to 2 hr collection
were replenished by the /3-alanine and malonate
added. Only trace amounts of ethylene were evolved
from samples that contained solubilized protein but
no substrates or cofactors, and substrate and cofactors alone evolved no ethylene.
Effect of Crude Enzyme Levels on Ethylene
Production. Collections were set up with ATP
(1.5 mM), 8-alanine and various weights of the
crude power up to 0.5 g (Fig. 3). No ethylene
was produced in the absence of protein. Ethylene
evolution over a 4 hr period was proportional to
enzyme
In
concentration
over
the
range
investigated.
separate experiment, 1.0 g of lyophilized powder
was found to evolve less ethylene than did 0.5 g,
a
under the same conditions as above.
Solubilization of the Particulate Enzyme System
and the Effects of Heating and Arsenite on the
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1220
~
PLANT PHYSIOLOGY
Table I. Cofactor and Substrate Stimulation of Ethylene Production by a Lyophilized Particulate Fraction
To 0.5g of the particulate fraction, the following were added: f-alanine (50 mM), malonate (50 mM), ci-ketoglutarate (50 mM), ATP (1.5 mM), CoA (0.17 mM), thiamine pyrophosphate (2.0 mM), MgSO4 (1.0 mM), and pyridoxal phosphate (0.5 mM). Collection was at pH 7.0 and room temperature. Final volume was 20 ml.
Expt
1
2
Collection period
0-2 hr
Sample
Ethylene
Stimulation
with cofactors
over powder
alone
Ethylene
with cofactors
over powder
alone
maUl
%
md
%
ul
25
z
_aA
zu
~115I-I
Lu
I0E 1
:a
8
In a
18.6
0
6.8
0
Powder +
substrate
and
cofactors
Powder
alone
25.6
+38
15.5
+128
26.4
0
10.2
0
Powder +
substrate
and
cofactors
31.5
+19
21.3
+109
,
.I
0.1
0
g
Stimulationi
Powder
alone
Soluble Sy,stem. The treatment involved a 10 hr
incubation of the crude enzyme at 00 with 0.4 %
Triton X-100 in 10 mm TES buffer, pH 7.6 (23).
This procedure was found to he superior to sonication, simple osmotic rupture, or treatment with 1 %
Tween 80. Triton X-100 at the 1 % level solubilized more protein than 0.4 % Triton X-100 but
was inhibitory to ethylene evolution. Treatment
with 0.4 % Triton X-100 solubilized approximately
50 % of the ethylene biosynthetic activity. The fact
that after 10 hr of incubation 50 % of the activity
still remained insoluble suggests -that at least part of
3
2-4 hr
.AI*
*
0.4
0.5
0.3
0.2
LYOPHILIZED PARTICULATE FRACTION
FIG. 3 3-alanine stimulated ethylene production over
a 4 hr period at various concentrations of lyophilized
subcellular particulate fraction. Reaction mixtures contained $-alanine (50 mM), ATP (1.5 mM), and TES
(50 mM), pH 7.0. Total volume was 20 ml.
the system may be associated with membranes of the
particulate components of the cell. The soluJble
enzyme system is unstable and becomes completely
inactive when stored for 24 hr at 0°.
When the soluble enzyme system was heated for
10 min in a boiling water bath, ethylene evolution
was inhibited 100 %, and when the system was
treated with 0.1 M sodium arsenite, ethylene production was inhibited 63 % (samples contained
9.7 mm dithiothreitol and substrates and cofactors
as given in table IT). Meheriuk (18) reported a
90 % inhibition of ethylene evolution from a tomato
particulate fraction with 0.1 M arsenite, but the
presence of dithiothreitol in the present samples
may have protected the enzymes.
Ethylene Biosynthesis bv Cytoplasmic Enzymes.
The supernatant layer from the. centrifugation at
32,000g to isolate the stbcellular fraction, contained
cytoplasmic enzymes. This cytoplasmic enzyme system was investigated for its ability to synthesize
ethylene from ,8-alanine alone, and in the presence of
soluibilized enzymes from the particulate fraction
(table II). The particulate fraction was by far the
better source of enzymes capable of synthesizing
ethylene. The data indicate that while the subcellular particulate fraction played a major role in
the biosynthesis of ethylene from fi-alanine, addition
of cvtoplasmic enzymes resulted in a 100 % increase
in evolution of the volatile. When the data were
converted to a specific activity (corrected for amount
of pro.tein), the soluTble particulate enzymes were
still superior to the cytoplasmic enzymes (table II).
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Copyright © 1969 American Society of Plant Biologists. All rights reserved.
STINSON AND
SPENCER--8-ALANINE
Table II. 3-alanine Stimulated Ethylene Biosynthesis
by Cytoplasmnic Enztymes Alone, antd in the Presence
of a Solubilized Particulate Fraction
Reaction mixture: 50 mM /-alanine, 50 mM malonate,
50 mm a-ketoglutarate, 0.83 mM NADH, 1.5 mm ATP,
2.0 mm thiamine pyrophosphate, 1.0 mM MgSO4, 0.5 mM
pyridoxal phosphate, 50 mm TES, pH 7.2. Final volume
was 20 ml. Protein was determined as outlined by Stinson and Spencer (21).
Enzyme
preparation
Sample
Cytoplasmic
1
enzymes
Particulate
fraction
Cytoplasmic
+ particulate
2
Cytoplasmic
enzymes
Particulate
fraction
Cytoplasmic
+ particulate
Collection period
0-4 hr
0- hr
Total
ethylene
Ethylene
evolved
/mg protein
102 X fl/Il
ni/Al
AS ANT
1221
ETHYLENE PRECURSOR
. A5
C
A
4
A
A
°
2340/
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¢ 1 t/^A 2-4h
A
0o
c
-~
1.5
0.5
0
2.5
3.5
4.5
B
6~~~~~~~~~
A
6.6
6.6
9.9
18.7
20.9
13.6
Z
2.9
2.1
tEi 24
~~~~
A
A
A
,.
h
~~~~~~AO0-2
3.9
8.6
11.5
6.4
0
10
20 30 40 50 60 70 80 90
mM
An enzyme essential for the conversion of f8-alanine
to ethylene may be limiting in the particulate system
bwecause this enzyme is localized primarily in the
cytoplasm. In a separate experiment. the cytoplasmic enzyme soltution was heated in a boiling water
bath for 10 min, then cooled, and solubilized particulate enzyme was added. The heated cytoplasmic
enzyme solution did not stimulate ethylene biosvnthesis in the soluble particulate enzyme system.
This finding indicated that the stimulatory factor in
the cytoplasmic fraction was heat labile, an,d there-
fore, possibly an enzyme.
f3-Alanine Levels anid Ethylene Biosynthesis by
the Solutble Enzyme System. Fig. 4 shows the
effects of various concentrations of /8-alanine on
ethylene biosynthesis by the solubilized particulate
fraction. This figure (A) indicates that evolution
of ethylene from both collections was almost maximal at 0.5 m-m /3-alanine. However, in a separate
experiment, the concentration range was 'extended
to 90 niM (B), and production of the volatile was
still increasing at this level of /3-alanine. It is
worth noting that the amount of ethylene evolved
from 0.5 mlm f-alanine represents a 0.002 % coInversion; this is the same conversion efficiency as
reported by Thompson and Spencer (27) when
f3-alanine-2-_4C was administered to a butanol treated
enzyme powder prepared from a subcellular particulate fraction of bean cotyledons. Obviously the
equilibria involved in the steps linking /3-alanine to
/3-ALANINE
FIG. 4. Ethylene biosynthesis by the soluble enzyme
system as a function of 3-alanine concentration. Reaction mixtures were 1.5 mM in ATP, 0.17 mm in CoA,
2.0 mm in thiamine pyrophosphate, 1.0 mm in MgSO4,
0.5 mm in pyridoxal phosphate, and 10 mm in TES, pH
7.0. GC'lections contained approximately 82 mg protein.
Final iolume was 20 ml.
ethylene do not favor ethylene production, but as
Thompson and Spencer (27) have shown, the postulated intermediates, Pl-hydroxypropionate and acrylate as well as ethylene become labeled when radioactive fl-alanine is added.
Ku and Pratt (10) with cell free fractions from
tonmatoes, and Burg and Clagett, (1) with banana
slices, were unable to stimulate ethylene evolution
with 3-alanine. However, Jacobsen and Wang (9)
and WAang et al. (29) report that the C-2 of fl-alanine
was heavily incorporated into -ethylene by Penicillium
but conclude that the pathway to ethylene
(ligitatuiniii
is
not direct.
excised
into
wax
-ethylene.
Knight (unpublished) found that
bean leaves converted 8-alanine-2-14C
Amino acid analyses of wax bean cotyledons
indicated the presence of 3-alanine, buit unknown
complicating factors did not allow quantitation.
Fowden i(4) reported that /8-alanine is widely distributed in plant tissues.
Malonate Levels and Ethylene Production From
the Soluble System. The pathway outlined by
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1222
PLANT PHYSIOLOGY
Thompson and Spencer (26) for the conversion of
,8-alanine to ethylene by a particulate enzyme system
involved malonate in a side path. They reported
that 50 mm malonate stimulated ethylene production
from fl-alanine by 83 % and suggested a precursor
role for this acid 1(27). Reduction of malonate to
malonate semialdehyde links the former with the
pathway that converts 8-alanine to ethylene. The
presence of both ,3-alanine and malonate resulted in
more ethylene than either alone. Fig. 5 shows that
ethylene evolution for the first 2 lhr period was
proportional to malonate concentration until -the acid
reached 35 m,M. Production of the volatile on the
2 to 4 hr collection at low malonate levels was
greater than on the 0 to 2 hr collection, but evolution
decreased at concentrations of malonate greater than
10 mM. This drop could be attributed to either
inhibitor accumulation or the depletion of an essential factor (NADH was not added to these samples
and therefore its depletion would not be responsible
for the decrease in evolution of the volatile on the
2 to 4 hr sampling). The largest stimulation (at
50 mM malonate) on the 0 to 2 hr collection was
2{04 % over a control with no malonate, whereas
maximum ethylene was evolved at 10 mM malonate on
the 2 to 4 hr collection and was 145 % over control.
It appears from Fig. 5 that malonate has a precursor
role, but this would be impossible to conform without
experiments with labeled substrates (see below).
Malonate may exert its effect as an end product
0
12
o 12
o
AO~~~~~~~~~~
E 1
4h
0 6
E
-C 5
"A
mN\ MALONIC ACID
I'IG. 5. Ethylene biosynthesis by the soluble enzyme
system as a function of malonic acid concentration. Reaction mixtures were 1.5 mM in ATP, 2.0 mM in thiamine pyrophosphate, 1.0 mM in MgSO4, 0.5 mM in
pyridoxal phosphate, and 10 mMi in TES, pH 7.0.
Approximately 82 mg of protein were in a final volumne
of 20 ml.
inhibitor, and prevent the 'flow' of carbons away
from malonate semial.dehyde. If this is the case,
then an ethylene precursor in the malonate reaction
mixture has yet to be identified.
To test whether malonate is, in fact, a precursor
of ethylene, a collection was set up that contained 1 ,uc
of malonate-2-'4C (3.4 ,uc/,umole). In addition the
sample contained enzyme, NADH (0.83 mM), ATP
(1.5 mM), thiamine pyrophosphate (2.0 mM),
MgSO4 (1.0 mM), and pyridoxal phosphate (0.5
IllM). Incubation was for 2 hr, and the radioactivity
in the hydrazone of malonate semialdehyde after
thin-layer chromatography was determined with a
Nuclear Chicago Unilux II liquid scintillation system. The RF values of the 2,4-dinitrophenyl hydrazone of malonate semialdehyde in (a) benzene:acetic
acid (19 :1), ('b) triethylamine :ether :water :pyridine
(60 :40:20 :20), and (c) ethanol :butanol :0.5 M ammonia (70:50:15) were 0.18, 0.50, and 0.33, respec-
tively.
No radioactivity could be detected in the silica
gel scrapings that corresponded to the 2,4-dinitrophenyl hydrazones of malonate semialdelhyde after
initial chromatography in benzene :acetic acid and
rechromatography in triethylamine :ether :water :pyridine, or in ethanol :bu'tanol :0.5 M ammonia. In
addition, no radioactivity could be detected in the
2.4-dinitrophenyl hydrazone of acetaldehyde, a decomposition product of the malonate semialdehyde
hydrazone. Very little ethylene was evolved and it
was not labeled. A separate experiment that contained 50 p.c of malonate-2-_4C (5 uc/pmole), NADH
(3.3 mM) and the amounts of ATP, thiamine pyrophosphate, MgSO4 and pyridoxal phosphate given
above, produced 31 m,ul of ethylene but no ethylene]4C. If the ethylene did originate from malonate
carbons, then label should have been incorporated
into the volatile. It appears that malonate is not an
ethylene precursor and its function in promoting
ethylene biosynthesis is one of preventing the loss of
carbons from the pathway.
What conipounds then form ethylene in the
malonate collections? Intermediates in the pathway
may be bound to enzymes, and the presence of
malonate 'forces' their conversion to ethylene. Also,
proteolytic enzymes and nucleic acid degrading
enzymes (28) may provide /8-alanine and other
amino acids to serve as ethylene precursors.
Ethylene Biosypnthesis as a Function of pH.
Fig. 6 shows that on both 0 to 2 and 2 to 4 hr
collections, ethylene producing activity reached a
peak at pH 7 and then declined. However, if the
pH was raised fur.ther, activity again increased for
both collection periods. Ethylene evolution on the
0 to 2 hr collection continued to rise, up to pH 12.
Evolution on the 2 to 4 hr collection reached a
second peak at pH 8.5, and then rapidly declined.
Although the 2 curves appear to be contradictory,
there may be a simple explanation; the large amounts
of ethylen-e evolved on the 0 to 2 hr collection at
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Copyright © 1969 American Society of Plant Biologists. All rights reserved.
1223
STINSON AND SPENCER--/3-ALANINE AS AN ETHYLENE PRECURSOR
I
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10
**A
20
30
40
50
60
70
s0
90
100
PROTEIN
FIG. 7. Ethylene production over
mg
a 4 hr period at
various concentrations of soluble enzyme solution. Reaction mixtures contained 13-alanine (50 mM), ATP (1.5
nmM), thiamine pyrophosphate (2.0 mM), MgSO4 (1.0
mM). pyridoxal phosphate (0.5 mM), NADH (0.83 mM),
and TES (50 mM), pH 7.2. Total volume was 20 ml.
Protein was determined as outlined by Stinson and
Spencer (21).
10
pH
FIG. 6. Effect of pH on ethylene biosynthesis by
the soluble enzyme system. Each assay contained f3-alanine (50 mM), malonate (50 mM), a-ketoglutarate (50
mM), ATP (1.5 mm), thiamine pyrophosphate (2.0 mM),
pyridoxal phosphate (0.5 mM), MgSO4 (1.0 mM), and
TES (10 mM). Approximately 82 mg of protein were
in a final volume of 20 ml.
pH 9 and above could cause a factor to become
limiting for the subsequent collection period.
It is significant that at pH 9 the cofactors alone
gave no ethylene. If the ethylene evolved from a
solution of protein alone *was collected for 2 hr at
pH 9 and then substrate and cofactors were added
to the sample, ethylene evolution increased several
hundred percent. This finding suggests that the
ethylene evolved at pH 9 may be enzymic but no
further experiments were conducted to ascertain if
any of the volatile was synthesized nonenzymically
at high pH. If the ethylene evolved at pH 10
(Fig. 6) and above is enzymic, it is unlikely that it
would be of significance in vivo. A maximum at
pH 7.0 simulates a more conventional pH dependence: Mapson and Wardale (16) found an enzyme
system from cauliflower florets that converted methional to ethylene maximally at pH 6.6, Shimokawa
and Kasai (21) found pH 6.0 was optimum for the
formation of ethylene from pyruvate by a subcellular
fraction from apple tissue, and Yang (30) found a
pH range from 7.3 to 8.3 was optimum for the conversion of methional to ethylene by horseradish
peroxidase. Ethylene evolution from biological systems at high pH values is not unnknown, Lieberman
et al. (14) reported increased production of the
volatile in apple tissue slices up to pH 9.5 and Ino
significan.t drop in evoltution to pH 10.5.
Levels of Soluble Protein and Ethylene Biosynthesis. Fig. 7 shows ethylene production over a
4 hr period at different enzyme concentrations.
Production was proportional to enzyme concentration
up to 15 mjul of ethylene and 45 mg protein/20 ml,
but beyond this production leveled off. It was interesting that evolution of the volatile over the
initial 2 hr period was proportional to enzyme concentration over the entire range investigated, but
evolution for the second 2 hr increased linearly only
up to 30 mg protein/20 ml, and then dropped sharply.
This suggests that some factor(s) are limiting on
the 2 to 4 hr collection.
Levels of NADPH anid NADH and Ethylene
Biosynthesis. The reductioln of malonate semialdehyde to 8-hydroxypropionate is a required reaction
in the conversion of fl-alanine to ethylene as proposed by Thompson and Spencer (26). However,
in their partially purified enzyme system from bean
cotyledons, the conversion occurred in the absence
of added reducing agent. Fig. 8 shows the amount
of ethylene evolved from the soluble enzyme system
when various levels of NADPH and NADH were
added. Both compounds greatly stimulated ethylene
production over a 2 hr collection, but NADH
achieved the same degree of stimulation at one half
the concentration of NADPH; maximum stimulation
for both reduced pyridine nucleotides was approximately 190 %. Comparisons were made only among
samples from the same enzyme preparation. NADH
and NADPH had their maximum effects on the
0 to 2 hr collection. Evolution on the 2 to 4 hr
collection was greater with NADPH (256 % stimulation at 1.12 mm) as opposed to only 160 % from
NADH). This may indicate that the large stimula-
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Copyright © 1969 American Society of Plant Biologists. All rights reserved.
1224
PLANT PH YSIOLOGY
16.5
A
A
15.0-
z
o 13.5
u
12.0
-
-
LU
10.5
-
LU
9.0
LU
75-5
z
,,06.0-
LU
Z
4.5
Lu
3.0-
LU
1.5
-
A
,
,
,
A NADH 0-2h
NADPH
e2t
system. Ethylene obtained in the absence of any reduced
pyridine nucleotide (control
5.5 m,ul) was subtracted
from that obtainled wvhenl reduced pyridine nucleotide
as added. Each collection was 50 mo in 3-alanine,
50 mI in maloniate, 50 im1Ni in a-ketoglutarate, 1.5 mm
in ATP, 2.0 mn\ inu thiamine pyrophosphate, 1.0 mM
in MgSO4, 0.15 une- in pyridoxal phosphate, pH 7.0.
F,inal volume of 20 ml conitained approximately 82 mg
of protein.
tioIn bx NADH on the () to 2 hr collectioni has nmade
another factor limitillg or has itself become exlhais-ted. The large aimilouint of NADH required to
obtaini miaxiimium ethylene evolution may indicate
(lestruction of the reduced pyridine nucleotide.
Fulrthier experimilents showed that none of 3-alanine.
a-ketoglutarate, or mlaloniate was limiting
on the
2 to 4 hr collection. However, when 12.5 mg NADH
were added after a 2 hr collection that initiallv
contained 12.5 mg NADH (0.83 m-m). an additional
stimu'lation of 88 % was achieved in 1 experiment
and 159 % with a differenlt enzyme preparation in a
second experimnent. These experiments prove only
that reducing equivalents are necessary for ethylene
biosynthesis in this s;ystem. Wlhen ,8-hydroxypropionate replaced ,8-alanine as substrate the requirenment for NADH was reduced but not eliminated;
this inidicates a possible inidirect role for the nucleotide.
Wlhen either dithiothreitol (9.7 nM) or NADPH
(0.56 -mM) was adde(d to a, suspension of 0.5 g
lyvophilized particulate fractioin that wvas 50 m-m in
,O-alanine, 50 nm.N in mnalonate, 50 mM in a-ketoglutarate, 10 nlAt In TES, and contained ATP,
tlhiaminie p)rophosp)hate, MgSO.1 (alnd pvridoxal phosphate I pH 7.0). there was nio augmientation of
evolved ethylene. However, when both compounds
were added together a 40 % stimulation in evolution
of the volatile was observed. Perhaps NADPH is
rapidly destroyed (oxidized) when ad,ded to the
crude system, and dithiothreitol is able to maintain
the pyridine nucleotide in a form (reduced) that can
be used by this system. The reduction of malonate
semia,ldehyde to 8-hydroxypropionate is a required
reaction in the conversion of ,(-alanine to ethylene
as proposed by Thompson and Spencer (26) and
NADPH or NADH may provide the reducing
equivalents.
Effect of a-ketoglutarate Addition oii Ethylene
Evolution. Thompson and Spencer (26) postulated
that the initial reaction in the conversion of f8-alanine
to ethylene was a transamination of the amino acid
-to malonate semialdehyde. When a-ketoglutarate
(50 mM) (a potential amino acce)tor for the aminotrainsferase), 3-alanine (50 m1M), anld cofactors were
added to a collection, these authors found that the
increase in ethylene evolution over a sample without
a-ketoglutarate, was 40 %. From these results it
was postulated that a-ketoglutarate was serving as
ani amino acceptor for a ,8-alanine aminotransferase.
Meheriuk, (18) obtained only a s'light stimulation
of ethylene generation wvhen a-ketoglutarate (1 mM)
was ad,ded to a tomato particulate fraction that was
50 m.m in fl-alanine. The present authors fouind
that when a-ketoglutarate ( 50 mMi) was added to
a solubilized particulate enzyme solution that contained malonate (50 mM) and f8-alanine (50 mM)
in the presence of cofactors, there was no change in
ethylene evolution on a 0 to 2 hr collection. When
a-ketoglutarate was added to samples that contained
the soluble enzyme preparation, cofactors, and either
malonate or /3-alanine, evolution of the volatile was
inhibited in both samples, but inhibition was much
greater in the reaction mixture that contained
,B-alanine. The a-ketoglutarate may stimulate the
drain of carbons from malonate semialdehyde to
acetyl units, a process that would be more prominent
xwith /3-alanine than with malonate as 'substrate'.
Postulated Intermiediates and Ethylene Biosynthesis. Several experiments in which various concentrations of the postulated intermediates were used
(plus cofactors as in Fig. 5) were averaged to
obtain the following amounts of ethylene evolved:
,3-alanine, 9.8 mli; malonate semialdehyde, 10.4
m,il; /3-hydroxypropionate, 11.2 m,ul; and acrylate,
10.9 m/11. The larger amounts of ethylene from
nialonate semialdehyde and /8-hydroxypropionate
than from /l-alanine, support the proposal that the
former 2 compounds are intermediates in the conversion of this amino acid to ethylene. Addition of
the more immediate ethylene precursor, acrylate,
resulted in more ethylene than /3-alanine but less
than /3-hydroxypropionate. Jacobsen and Wang (9)
reported -that Penicillium digitatitm preferentially
incorporates the C-2 of acrylate into ethyilene and
suggest that acrylic acid is an immediate precursor.
Thompson 1(25) reported that 75 mm acrylate in-
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Copyright © 1969 American Society of Plant Biologists. All rights reserved.
STINSON AND SPENCER-/8-ALANINE AS AN ETHYLENE PRECURSOR
hibited evolution of the volatile by 70 % and speculated that the extreme reactivity of the compound
allows it to attack other components, such as essential sulfhydryl groups of enzymes, in the reaction
mixture. Thijsse (24) has reported that acrylate
(14 mM) inhibited -the a8-oxidation of lipids (,8-oxidation is involved in the formation of ethylene from
,8-alanine). Experiments of the present authors
conducted in the presence of dithiothreitol (9.7 mM),
a sulfhydryl protector (3), showed that this dithiol
could increase the stimulation achieved with acrylate
3.5 fold.
Incorporationt of Tritium From Tritiated Water
Into Ethylene. Examination of the scheme for the
conversion of /8-alanine to ethylene reveals that the
water medium could play an important role. If the
decarboxylation of acrylate involves thiamine pyrophosphate as in the generally accepted mechanism,
then 2 protons are incorporated into the ethylene
molecule, 1 on each carbon atonm. If there is an
equilibrium between each step and the proton source
is tritiated water, it is possible for all 4 'hydrogens',
of the ethylene molecule to be labeled. In addition,
the dehydration and redox steps involved in the
postulated pathway could allow for the incorporation
of tritium. To see if tritium from tritiated water
could 'be incorporated into ethylene, a sample was
set up as usual except that the protein solution was
freeze-dried to reduce the final volume to 8.0 ml.
The sample contained malonate, /3-alanine, the usual
cofactors, 150 mc 3H20 !(0.338 jxc/,nmole), and TES
buffer, pH 7.2. Ethylene collections were made for
0 to 16 hr and 16 to 23 hr. Radioactivity in the
ethylene was determined by gas radiochromatography.
Ethylene-3H was not available to calibrate the instrument; an efficiency one lhalf of that obtained for
ethylene-'4C was assumed.
Table III shows the results of a tritium experiment; radioactive ethylene was detected in both
collection periods. The specific activity of the
evolved ethylene (table I) doubled on the second
collection period, and thus, the ratio of the specific
Table III. Incorporation of Tritiumn From Tritiated
Water Into Ethylene
Collec-
Ethylene
specific
Specific activity
ethylene:
specific activity
tion
period Ethylene Ethylene radio activity
3H20
hr
dpm
ratio
Ac/lmole
m/Al
0-16
90
970
0.110
0.33
16-23
25
480
0.196
0.60
Reaction mixture was 60 mm in 1-alanine, 60 mM
in malonate, 4.0 mm in NADH, 1.8 mM in ATP, 2.4
mm in TPP, 1.2 mm in MgSO4, 0.6 mm in pyridoxal
phosphate, and 60 mM in TES, pH 7.2. Final volume
was 8 ml and contained 150 mc 3H,O (0.338 pc//Amole).
The reaction mixture contained approximately 82 mg of
protein.
1225
activities of ethylene-3H and 3H2O also increased.
This increased specific activity may indicate either
the influence of bound intermediates or greater
operation of a pathway, other than the /8-hydroxypropionate route, 'during the first collection period.
The finding by Burg and Thimann (2) that tritium
from 3HI2O was rapidly incorporated into ethylene
suggested to them that one of the terminal steps in
the hiosynthesis may be a dehvdration. This finding
and subsequent postulation are in agreement with
the data of table III, and are in accordance with the
proposed scheme for the conversion of /3-alanine to
ethylene.
Conclusions
The results reported here, wvhen coupled with
those of Thompson and Spencer (26, 27), leave little
doubt that bean cotyledons contain enzymes capable
of generating ethylene from /8-alanine. In addition,
the demonstration by S'tinson and Spencer (23) of
the presence of an aminotransferase that converts
/8-alanine to malonate semialdehyde in the solubilized
particulate enzyme system further supports this
proposal.
Literature Cited
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125-30.
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PLANT PHYSIOLOGY
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