Plant Physiol. 1974 Beale 291 6
Plant Physiol. 1974 Beale 291 6
Plant Physiol. 1974 Beale 291 6
form-October 5, 1973
is thought to occur before ALA formation because: (a) no intermediates other than protochlorophyllide accumulate when
greening is inhibited by placing plants in the dark; and (b)
exogenous ALA is converted to protochlorophyllide in plants
kept in the dark, and a portion of this ALA-induced protochlorophyllide can be converted to Chl during subsequent incubation of the plant in the light (16, 20).
In animal tissues, bacteria, and yeast, ALA is formed by the
condensation of succinyl CoA and glycine, catalyzed by the
pyridoxal phosphate-requiring enzyme succinyl CoA-glycine
succinyl transferase (ALA synthetase) yielding a-amino, ,Bketoadipic acid, which is immediately decarboxylated to ALA
and CO2 (derived from the carboxyl carbon of glycine) (11).
In photosynthetic bacteria and in some heme-forming systems,
ALA synthetase is a site of regulation of the pathway, being
subject to feedback inhibition and induction (5, 12).
The evidence for the presence of ALA synthetase in greening plant tissue is very weak. Although the enzyme would be
expected to be very active during the rapid phase of Chl synthesis in greening etiolated tissues, it has not been detected in
extracts from such material. Where ALA synthetase has been
reported in plant sources, the evidence for its existence has
been fragmentary, and the relation of the enzyme to Chl synthesis remains undetermined (14, 17, 24, 25).
In this study we have concerned ourselves with the following
questions: (a) whether ALA is indeed synthesized by greening
plant tissues; (b) if so, whether this ALA is the precursor of
the tetrapyrrole moiety of Chl; (c) whether the formation of
ALA is the rate-limiting step in the biosynthetic pathway leading to Chl in greening plant tissues.
In these experiments we have used the ALA-dehydrase inhibitor levulinic acid to induce the accumulation of ALA in
greening plant tissues. This technique was originated by Beale
(2, 3) to demonstrate the biosynthesis of ALA in the unicellular alga Chlorella and was subsequently used for the same
purpose in bacteria (10), Euglena (18), corn, and bean leaves
(9). In a companion paper, we describe an inquiry into the in
vivo source of this ALA (4).
Reagents. Levulinic acid and Ehrlich's reagent (p-dimethylaminobenzaldehyde) were purchased from Sigma Chemical
Co., dimethylsulfoxide from J. T. Baker, ethylacetoacetate and
acetyl acetone (2, 4-pentanedione) from Matheson Coleman
and Bell, and Botran (2,6-dichloro-4-nitroaniline) from Duco
Products Co., division of Upjohn Co., Kalamazoo, Mich.
DCMU, obtained from E. I. duPont de Nemours and Co.,
was recrystalized from aqueous acetone.
Plant Material. Cucumber seeds (Cucumis sativus L. var.
Alpha green), a gift of the Niagara Chemical Division, FMC
Corporation, Modesto, Calif. were germinated in complete
292
53, 1974
Concentration Effects of Levulinic Acid. Six-day-old etiolated cucumber cotyledons were incubated at 28 C in the light.
After 4.5 hr, samples were treated with various concentrations
of levulinic acid and allowed to incubate for 2 more hours,
then the tissue was homogenized, and the accumulated ALA
was determined (Table I). Because 100 mM levulinic acid gave
the highest yields of ALA, it was chosen as the standard concentration in subsequent experiments with cucumber cotyledons.
Correlation of Rates of ALA and Chl Synthesis. Etiolated
cucumber cotyledons were incubated in the light. and samples
were taken for periodic Chl determination. Other samples
were withdrawn at regular intervals and incubated with 100
mM levulinic acid for 1 hr in the light, after which the accumulated ALA was determined. The rates of Chl synthesis and
ALA accumulation are plotted (Fig. 1). A close relationship
between shapes of the resulting curves is observed over the
first 8 hr of the greening process.
Effect of Returning Tissue to Darkness. Etiolated cucumber
cotyledons were illuminated for 4.5 hr, then were treated
with 100 mm levulinic acid. After 1 additional hr in the light,
some of the samples were placed in the dark at 25 C. Samples
were taken at regular intervals and the accumulated ALA was
determined (Fig. 2). ALA accumulation stopped within 1 hr
after the tissue was returned to the dark.
Stoichiometry of ALA and Chl Formation. Etiolated cucumber cotyledons were illuminated for 4.5 hr, then some samples
were treated with 100 mm levulinic acid and others were left
untreated. After 2 additional hr, both Chl and accumulated
ALA were measured in all samples. One hundred mm levulinic
acid was found to inhibit Chl synthesis by 50% under these
conditions, but the total ALA synthesized (moles ALA accumulated plus 8 times moles of Chl formed) in the levulinic
acid-treated tissue approximated that of the controls (Table
II).
Effect of Metabolic Inhibitors and Anaerobiosis. Etiolated
cucumber cotyledons were illuminated for 4.5 hr, then treated
with 100 mm levulinic acid and one of the inhibitors listed
in Table III, and allowed to incubate for 2 more hours, after
which the ALA was determined and compared to a sample
treated with levulinic acid only. In the NM-treated sample, N.
was allowed to flush a covered Petri dish containing the
cotyledons. This sample was compared to a similar one flushed
with air. Anaerobiosis inhibited ALA accumulation almost
completely, azide and cyanide inhibited by about 70% at the
concentrations employed, malonate and arsenite showed a
moderate but appreciable inhibition, whereas the other inhibitors had a relatively small effect or none at all (Table III).
The inhibitions caused by anaerobiosis or KCN could not be
reversed by the addition of 100 mm a-ketoglutarate, 100 mM
glycine, and 10 mm NAD. Furthermore, the inhibition caused
ALA ACCUMULATION
293
TISSUES
TO LEVULINIC ACID
Barley. Seven-day-old etiolated barley seedlings were illuminated intact for 4 hr at 30 C, then the primary leaves were
cut into 1-cm segments and allowed to float in Petri dishes containing 2.5 ml of solution of levulinic acid. After 3 additional
Table I. ALA Accumulationi in Greeninlg Cucumber Cotyledoiis hr of incubation, the leaf segments were homogenized in 5%
HClO, and ALA was determined. Twenty mm levulinic acid
Treated with Levulinzic Acid
was found to cause the higher ALA accumulation of the two
Six-day-old etiolated cucumber cotyledons were preilluminated
for 4.5 hr, then illumination was continued for 2 more hr in the concentrations tested (Table IV).
Chl synthesis was inhibited by both concentrations of levupresence of 10% dimethylsulfoxide and the indicated concentralinic acid; but in the presence of 100 mm levulinic acid a net
tions of levulinic acid.
loss of Chl from the tissue occurred. At either concentration
levulinic acid caused a marked inhibition of the total ALA
ALA Accumulated in 2 H r
Levulinic Acid Concn
(moles ALA plus 8 times moles Chl) accumulated; while with
m?lM
nrnoles,'g fresh ut
cucumber cotyledons the ALA and the Chl were essentially
0
0
additive even with 100 mm levulinic acid (compare Tables II
7.8
1
and IV).
39.4
10
Beans. Ten-day-old etiolated bean seedlings were illumi107.0
25
nated for 5 hr at 28 C, then the primary leaves were excised,
121.0
50
separated, and placed in Petri dishes, along with 2.5 ml of
173.3
100
levulinic
acid solution. After 3 more hr of illumination, the
138.6
1000
tissue
was
homogenized and ALA was determined. ALA
62.9
3000
accumulation increased with increasing levulinic acid con0.0
10000
centration up to 20 mM, and then reached a plateau (Table V).
130
110
aI
0
30
10
FIG. 1. Correlation of Chl formation and ALA accumulation in greening cucumber cotyledons. Six-day-old etiolated cucumber cotyledons
were illuminated with 240 ft-c of fluorescent light at 28 C. Periodic samples were taken for Chl determinations. Other samples were taken. incubated with 100 mm levulinic acid and 10c dimethylsulfoxide for 1 hr under the same light and temperature conditions as above, and then accumulated ALA was determined. Amounts of ALA accumulated/hr- g tissue in treated samples (ALA) and Chl formed/hr- g tissue in untreated samples (CHL) are both plotted against hours since beginning of illumination.
Downloaded from www.plantphysiol.org on May 28, 2014 - Published by www.plant.org
Copyright 1974 American Society of Plant Biologists. All rights reserved.
294
from succinyl CoA and glycine has been found to be the site of
regulation of the pathway. An exception to this general finding
may exist in Neurospora, where it has been reported that the
second enzyme in the pathway, ALA dehydrase, is the site of
regulation (15).
Table III. Inihibitioni of Levulilnic Acid-inzduced ALA Accimuilation
in Cucumber Cotyledonis by Various Substanices
Etiolated cotyledons were preilluminated for 4.5 hr, then illumination was continued for 2 hr in the presence of 10%O dimethylsulfoxide, 100 mm levulinic acid, and the inhibitor, applied by
wetting the cotyledons (N2-treated samples were flushed with a
constant stream of the gas).
Chemical
N2 (100%7C)
KCN
KCN
2,4-Dinitrophenol
Na
Na
Na
Na
Na
Na
malonate
malonate
arsenite
arsenite
fluoroacetate
fluoroacetate
Hydroxylamine-HCI
Hydroxylamine-HCl
0
HOURS
AFTER
ADMINISTRATION OF
4
100 mM LEVULINIC ACID
Experiment
Sample
Chl
formed
in 2 hr
and C)
II
87.1
697
100 mM levulinic
44.7
357
0
269
697
626
acid
Control
100 mM levulinic
acid
59.0
25.6
472
205
0
303
472
508
DISCUSSION
The regulation of a biochemical pathway is generally considered to be exerted most effectively at the enzymatic step
which produces the first compound unique to that pathway. Chl
and heme are thought to share a common biosynthetic pathway
from ALA to protoporphyrin IX (7). In studies on heme synthesis in mammalian (12) and bacterial (22) systems, and of
bacteriochlorophyll synthesis (5, 23), the formation of ALA
1
3
1
10
100
0.1
10
0.3
3
0.3
3
0.3
3
1
90; 87
72
70
24
0
37
17
35
11
0
8
2
9
66
2
Levulinic Acid
Concn
Chl
formed
in 3 hr
Control
C
ITotal ALA
ALA acEight
cumulated
times
in 3 hr
column A
Synthesis
(sum of B
+ C)
Initonf
Inhibition of
Total ALA
Synthesis
nmoles/g fresh wt
MM
20 mM
100 mM
Control
Inhibition
Total ALA
ISynthesis
(sum of B
ALA acEight
cumulated'
times
column a in 2 hr
nmoles/g fresh wi
NaN3
NaN3
DCMU
Concn
140
61
(-7.3)1
1120
488
-58
0
289
250
1120
777
192
31
83
ALA Accumulated in 3 hr
mM
nmoles/g freslh wt
0
40
114
212
201
207
5
10
20
40
100
ALA ACCUMULATION
The formation of ALA is generally thought to be the limiting step in the pathway leading to protochlorophyllide in
etiolated higher plant tissue. This conclusion is based primarily on experiments wherein ALA fed to the etiolated plant
tissues results in higher protochlorophyllide content than in
control tissues. At least some of this ALA-protochlorophyllide
can be photoconverted to chlorophyll(ide) when the etiolated
tissue is illuminated (16, 20).
Presumably, the enzymes involved in the pathway from ALA
to protochlorophyllide are present in the etiolated tissue in
nonlimiting quantities, but it is not presently known whether
in general these enzymes are constitutive or if they are induced
during the course of greening. Steer and Gibbs (21) were able
to demonstrate changes in ALA dehydrase activity in greening
bean leaves, but these changes were not sufficiently large to
indicate a regulatory function for this enzyme. Our finding
that ALA dehydrase activity is maintained at a constant level
as etiolated cucumber cotyledons are allowed to become green,
supports the hypothesis that this enzyme is not involved in
the regulation of the Chl pathway.
Although the enzyme ALA synthetase (succinyl CoA-glycine
succinyl transferase) has been reported to occur in crude plant
extracts, the reports have been either preliminary accounts
or abstracts not followed by more comprehensive confirmatory
studies (14, 24) or they are subject to the following criticisms:
(a) lack of correlation of enzyme activity with changing rates
of Chl synthesis; (b) lack of the crucial test for succinyl
transferase activity which is the incorporation into ALA of the
methylene carbon of glycine but exclusion of the carboxyl
carbon; (c) lack of adequate precautions against microbial contamination during the preparation or incubation of the extracts (17, 25).
It has recently become possible to measure ALA formation
in vivo by inhibiting ALA dehydrase with the competitive inhibitor levulinic acid and measuring the accumulation of ALA
as it is formed (2, 3). A potential disadvantage of this method
is the possibility of other effects of levulinic acid on cell
metabolism, and therefore of secondary effects on ALA accumulation. An awareness of this possibility leads us to seek
converging lines of evidence from experiments with different
plant tissues and experimental conditions.
We have shown here that the time course of ALA accumulation in greening cucumber cotyledons treated with levulinic
acid corresponds to the time course of Chl formation in control tissue (Fig. 1), confirming similar findings in Chlorella (2)
and corn (9). Light is required for ALA accumulation in
levulinic acid-treated cucumber cotyledons (Fig. 2) as it is
in other higher plants (9) and in Euglena (18). In all these
cases there is a corresponding light requirement for Chl
accumulation in control tissues.
In growing Chlorella cultures, it was shown that the total
quantity of ALA synthesized, that is, the moles of accumulated ALA plus 8 times the moles of Chl formed (since 8
molecules of ALA are required to form 1 of Chl), was the
same in levulinic acid-treated cultures as in untreated controls,
even when Chl synthesis was inhibited 50%. This finding provided strong evidence that the ALA which accumulated in the
presence of levulinic acid was indeed destined for Chl synthesis and that the levulinic acid was without side-effect,
serving only to divert ALA from the Chl pathway. This
stoichiometry has now been demonstrated in cucumber cotyledons (Table II). However, in barley leaves, this could not be
shown. Levulinic acid, at a concentration sufficient to cause
accumulation of free ALA, inhibited the formation of total
ALA (Table IV). It is interesting that in long term incubations
of bean and corn leaves employed by Harel and Klein (9),
295
L. Ginzton for
performing the
chlorophyll deter-
minations.
LITERATURE CITED
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Plant Physiol. 45: 504-506.
3. BEALE, S. I. 1971. Studies on the biosynthesis and metabolism of 3-aminolevulinic acid in Chlorella. Plant Physiol. 48: 316-319.
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7. GRANXICK, S. AND S. SASSA. 1971.
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19. SCHIFF, J. A., MI. H. ZELDIN, AND J. RUBIMAN. 1967. Chlorophyll formation
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