Plant Physiol. (1990) 94, 1365-1375
0032-0889/90/94/1365/1 1/$01 .00/0
Received for publication May 14, 1990
Accepted July 9, 1990
Light Regulation of b-Aminolevulinic Acid Biosynthetic
Enzymes and tRNA in Euglena gracilis'
Sandra M. Mayer and Samuel 1. Beale*
Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912
ABSTRACT
biosynthesis of the chloroplast tetrapyrrole pigment precursor,
ALA2, from glutamate via the tRNA-dependent five-carbon
pathway (24), rather than by condensation of glycine and
succinyl-CoA, catalyzed by ALA synthase, which occurs in
animal cells, fungi, and some bacteria. The five-carbon pathway is proposed to begin with activation of glutamate by
ligation to tRNAG"U, followed by reduction of the activated
glutamate to GSA or a closely related molecule, and transamination of GSA to yield ALA (Fig. 1). In vitro ALA-forming
extracts from barley (8), Chlorella (40), and Synechocystis
(28) have been separated into three enzyme fractions and
tRNAGlU, all ofwhich are required, along with ATP, NADPH,
and Mg2", for ALA formation from glutamate.
Experiments with inhibitors of ALA utilization have shown
that, in both Euglena cells (27) and plant tissues (4), ALA
formation is the rate-limiting step of pigment accumulation
during the greening of dark-grown cells and tissues when
exposed to light. It has also been shown that, in both Euglena
and plants, cycloheximide-sensitive protein synthesis on cytoplasmic ribosomes is required for greening to occur (21,
25), suggesting that light induces the synthesis of one or more
of the enzyme components of the five-carbon ALA biosynthetic pathway.
Euglena is known to have two photoregulatory systems.
One of these responds to both red and blue light, appears to
be localized in the plastids, is lacking in certain mutant strains
having nonfunctional or absent plastids, and is thought to
have protochlorophyllide as its photoreceptor (10, 33). The
other photoregulatory system responds only to blue light and
retains its function in mutant cells lacking functional plastids
(30, 33). Neither phytochrome nor phytochrome responses
have been reported in Euglena.
In this report, we have investigated the role of light in
modulating cellular levels of the tRNA and the three enzyme
reactions required for ALA formation from glutamate in
Euglena extracts. Our results indicate that, in Euglena, cellular levels of both the tRNA and the enzymes are regulated
by light, that the five-carbon ALA-forming pathway is induced
by both red and blue light, and that the rate-limiting component in dark-grown cells may be GSA aminotransferase.
2Abbreviations: ALA, 6-aminolevulinic acid; AHA, 4-amino-5-
Chlorophyll synthesis in Euglena, as in higher plants, occurs
only in the light. The key chlorophyll precursor, 6-aminolevulinic
acid (ALA), is formed in Euglena, as in plants, from glutamate in
a reaction sequence catalyzed by three enzymes and requiring
tRNAGlU. ALA formation from glutamate occurs in extracts of lightgrown Euglena cells, but activity is very low in dark-grown cell
extracts. Cells grown in either red (650-700 nanometers) or blue
(400-480 nanometers) light yielded in vitro activity, but neither
red nor blue light alone induced activity as high as that induced
by white light or red and blue light together, at equal total fluence
rates. Levels of the individual enzymes and the required tRNA
were measured in cell extracts of light- and dark-grown cells.
tRNA capable of being charged with glutamate was approximately
equally abundant in extracts of light- and dark-grown cells. tRNA
capable of supporting ALA synthesis was approximately three
times more abundant in extracts of light-grown cells than in darkgrown cell extracts. Total glutamyl-tRNA synthetase activity was
nearly twice as high in extracts of light-grown cells as in darkgrown cell extracts. However, extracts of both light- and darkgrown cells were able to charge tRNAGlu isolated from light-grown
cells to form glutamyl-tRNA that could function as substrate for
ALA synthesis. Glutamyl-tRNA reductase, which catalyzes pyridine nucleotide-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde (GSA), was approximately fourfold greater
in extracts of light-grown cells than in dark-grown cell extracts.
GSA aminotransferase activity was detectable only in extracts of
light-grown cells. These results indicate that both the tRNA and
enzymes required for ALA synthesis from glutamate are regulated
by light in Euglena. The results further suggest that ALA formation
from glutamate in dark-grown Euglena cells may be limited by the
absence of GSA aminotransferase activity.
The phytoflagellate Euglena gracilis shares with higher
plants a light requirement for Chl synthesis and the development of photosynthetic competence (33). As with plant tissues, Euglena cells grown in complete darkness fail to accumulate Chl, and instead form small quantities of Pchl(ide).
Exposure to light brings about the reduction of these pigments
to Chl(ide), and in continuous light this is followed by a phase
of rapid Chl accumulation (17) and transformation of developing plastids into photosynthetically functional chloroplasts
(34).
Another feature of Euglena that is shared with plants is
hexynoic (acetylenic-GABA) acid; gabaculine, 3-amino-2,3-dihydrobenzoic acid; GSA, glutamate-l-semialdehyde; PALP, pyridoxal-5phosphate; RNasin, human placental RNase inhibitor; BU, Blue
Sepharose unbound protein fraction.
'Supported by National Science Foundation grant DMB85- 18580.
1365
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1 366
COOH
0H2
MAYER AND BEALE
COOH
CH2
ATP, Mg2,OCH2- tRNAGlU,
CH2
Glutamyl-tRNA Synthetase
CHNH2
COOH
Glutamic Acid
COOH
COOH
CH2
NADPH
Plant Physiol. Vol. 94, 1990
CH2
PALP
-e- CH2
AminotransferaseI
Glutamyl-tRNA'-CH2 GSA
CHNH2
Reductase
CHNH2
C=O
C=O
C=O
tRNAGIU
H
Glutamyl-tRNA
GSA
MATERIALS AND METHODS
Growth of Cells
Axenic cultures of Euglena gracilis Klebs var Pringsheim
strain Z (originally obtained from H. Lyman, State University
of New York, Stony Brook) were grown in glucose-based
heterotrophic medium as previously described (5). In all experiments, cell population doubling times were within the
range 10 to 13 h. Cultures referred to as light-grown were
grown in liquid medium with rotary shaking at 25°C at an
incident light intensity of 32 ME m-2 s-1 supplied by equal
numbers of red and cool-white fluorescent tubes. Cells referred to as dark-grown had been subcultured in continuous
darkness for at least one year prior to experiments.
Cells grown for experiments in which the effects of spectral
regions of light were being studied were grown in liquid
medium, with shaking, in an atmosphere of 5% (v/v) 02 in
N2, in chambers constructed to allow illumination of the
cultures with spectrally filtered light. Light intensities were
measured with a LI-COR model LI-185A quantum radiometer/photometer, equipped with a model LI-190S quantum
sensor, which has an equalized response within the range 400
to 700 nm. In these experiments, the fluence rates through all
filters were equalized to 13 ME m-2 s-' by supplementation of
the light available from the fluorescent tubes with a tungsten
halogen lamp, or by interposing neutral density filters consisting of Miracloth light-scattering fabric. Kodak Wratten
filters No. 98 Blue (CAT 174 0893), No. 70 Red (CAT 149
7668), or a hybrid filter with half the filter area No. 98 Blue
and the other half No. 70 Red were interposed between the
light source and the cultures. The red filter transmitted light
only above 650 nm, and the blue filter transmitted light only
between 400 and 480 nm (plus a small amount of light above
720 nm; see "Results").
Cell Extraction for Enzyme Preparation
Cell cultures in exponential growth phase were thoroughly
chilled on ice under the light conditions in which they were
grown. All subsequent operations were performed at 0 to 4°C.
Cells were harvested by centrifugation, washed, resuspended
in two volumes of homogenization medium (200 mm glycerol,
100 mm Tricine, 15 mM MgCl2, 1.0 mm DTT, 20 Mm PALP,
0.004% PMSF [pH 7.9]), and disrupted by sonication. Large
volumes of cells were disrupted by sixteen 30-s sonic bursts
separated by 30-s cooling periods; small volumes were sonicated by ten 20-s bursts separated by 30-s cooling periods,
using a rosette sonication vessel (Heat Systems-Ultrasonics,
Farmingdale, NY) immersed in ice. Cell debris and unbroken
CH2NH2
Figure 1. Proposed route of ALA formation from
glutamate.
ALA
cells were removed by centrifugation for 10 min at 10,000g.
The supernatant was adjusted to 0.5 M NaCl and stirred for
20 min, then clarified by centrifugation for 90 min at
264,000g, and desalted by gel filtration through Sephadex G25, as previously described (24). The desalted supernatant was
fractionated by differential (NH4)2SO4 precipitation between
35 and 60% of saturating concentration of (NH4)2SO4, in the
presence of 5 mm EDTA and 0.004% PMSF, desalted by
passage through Sephadex G-25 that was preequilibrated with
column buffer (1.0 M glycerol, 50 mM Tricine, 15 mM MgC92,
1.0 mM DTT, 20,M PALP [pH 7.9]), then eluted with column
buffer containing 0.004% PMSF, and stored at -75°C. In
some cases, where indicated, the differential (NH4)2SO4 precipitation step was omitted.
Enzyme Fractionation by Affinity Chromatography
Fractionation of extracts from light-grown cells on Reactive
Blue 2-Sepharose was carried out as previously described (40).
A portion of the desalted 35 to 60% (NH4)2SO4 fraction
described above, representing approximately 20 g of cells, was
diluted to 50 mL with column buffer and applied to a 4.7 cm
long x 2.5 cm diameter column ofReactive Blue 2-Sepharose.
The portion of the extract that did not bind to the matrix was
eluted with approximately 250 mL of column buffer (without
added salt) until the protein content was barely detectable by
the dye-binding assay (7). This fraction, designated BU, was
precipitated with (NH4)2SO4 at 70% of saturation, dissolved
in column buffer, desalted, and stored at -75C.
Cell Extraction for tRNA
The procedure employed was similar to those previously
described for isolation of tRNA from Euglena (12) and Chlorella (37). RNA was prepared by extraction of high-speed
supernatant obtained from cells sonicated in RNA extraction
medium (100 mM NaCl, 10 mM Tris-HCl, 10 mm Mg-acetate,
10 mm f3-mercaptoethanol, 5 mM EDTA [pH 7.5]) at a ratio
of 2 mL of buffer per g of cells. The supernatant was adjusted
to a volume of 4 mL per g of cells, SDS was added to a final
concentration of 1 % (w/v), and the solution was mixed with
an equal volume of phenol (previously equilibrated with RNA
extraction medium). The separated phenol phase was extracted with 0.5 volume of RNA extraction medium, the
aqueous phases were combined, 2.5 volumes of absolute
ethanol were added, and the nucleic acids were precipitated
overnight at -20°C, collected by centrifugation, dissolved in
RNA extraction medium, and extracted three to four times
with equal volumes of chloroform:iso-amyl alcohol (24: 1, v/
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LIGHT REGULATION OF ALA BIOSYNTHETIC STEPS IN EUGLENA
v). tRNA was isolated by DEAE-cellulose chromatography as
previously described (1). tRNA was deacylated by dissolving
the precipitate in 500 mm Tris-HCI (pH 8.0) and incubating
at room temperature for 2 h. The deacylated RNA was
precipitated by the addition of one-tenth volume of 20% (w/
v) Na-acetate and 2.5 volumes of absolute ethanol and cooling
to -75°C, collected by centrifugation, washed with 70%
ethanol, and dried with a stream of air while maintained at
0°C on ice. The tRNA was redissolved in extraction medium
containing 1.0 mm DTT and stored in small aliquots at
-20°C. In later preparations, cells were harvested and sonicated as described above, then stirred with phenol and SDS
without prior centrifugation, and tRNA was isolated as described above.
Colorimetric Assay for in Vitro ALA Formation
Assays were carried out by modifications of a previously
described method (38). Incubation was for 60 min at 30°C in
0.25 to 1.0 mL of assay medium (1.0 M glycerol, 50 mm
Tricine, 15 mM MgCl2, 5 mM ATP, 5 mM levulinic acid, 1.0
mM NADPH, 1.0 mm glutamate, 1.0 mM DTT, 20 uM PALP
[pH 7.9]). Incubations contained 10 A260 units of RNA and
2.0 to 5.0 mg of enzyme protein per mL of reaction volume.
Reactions were terminated by addition of one-tenth volume
of 1.0 M citric acid and one volume of 10% (w/v) SDS
followed by heating for 3 min at 95°C. ALA was isolated on
Dowex 50W-X8 (Na), ethylacetoacetate was added, and the
solutions were heated to 95°C for 15 min to form 1-methyl2-carboxyethyl-3-propionic acid pyrrole. The product was
quantitated spectrophotometrically after reaction with an
equal volume of Ehrlich-Hg reagent, using a Cary model 219
spectrophotometer (Varian). The A553 of unincubated control
samples was subtracted from those of incubated samples to
determine net A553 values, and ALA was calculated from a
standard curve with samples containing known amounts of
ALA.
Assay for Glutamate Accepting Ability of tRNA
['4C]Glutamyl-tRNA formation from ['4C]glutamate was
measured by appearance of TCA-precipitable radioactivity
(24). Incubation was carried out for 30 min at 30°C in 250
,uL of reaction medium similar to the assay medium for ALA
formation. NADPH and levulinic acid were omitted from
this medium, and the glutamate was present at a concentration of 100 jM and contained 840,000 cpm of ['4C]glutamate
(the final specific radioactivity was 34 cpm pmol-'). Reactions
were terminated by the addition of 1.0 mL of 84% (v/v)
aqueous acetone containing 12.5% (w/v) TCA. After standing
on ice for 15 min, the mixtures were centrifuged for 2 min at
13,500g in an Eppendorf microcentrifuge. The pellets were
washed once with 1.5 mL of 67% (v/v) aqueous acetone
containing 10% (w/v) TCA and 1.0 mm glutamate, then twice
with 1.5-mL portions of 10% (w/v) aqueous TCA containing
1.0 mM glutamate, and finally with 1.0 mL of 95% (v/v)
aqueous ethanol. The final pellets were dissolved in 100 ,L
of 88% (w/v) aqueous HCOOH, 1.7 mL of Tritosol (14)
liquid scintillation solution was added, and radioactivity was
determined by liquid scintillation spectroscopy in a Beckman
1 367
LS-1OOC instrument. Zero-time controls had fewer than 200
cpm in the final pellets, and all duplicates agreed within 15%.
Glutamyl-tRNA Synthetase Assay
A tritium-based filter binding assay similar to that previously described was used (31). Reactions were carried out in
500 .L of column buffer containing 5 mm ATP, 25 A260 units
of tRNA isolated (as described above) from light-grown cells,
10 gM final glutamate concentration having 3.3 x IO' cpm of
[3,4-3H]glutamate (the final specific radioactivity was 6600
cpm pmol-'), and enzyme extract (600 ,ug of protein). Enzyme
extract was prepared as a 35 to 60% differential (NH4)2SO4
precipitate (as described above) from cells grown in the light
or dark. The reactions were initiated by addition of enzyme
that was prewarmed to 30°C, incubation was for 5 min at
30°C, and the reactions were terminated by the application of
1 5-,uL samples onto glass fiber filters, which were immediately
immersed in ice-cold 10% (w/v) aqueous TCA containing
100 mm unlabeled glutamate. Filters were washed extensively
with cold 5% (w/v) aqueous TCA, then with ethanol:ethyl
ether (1:1, v/v), and finally with ethyl ether as previously
described (31), air dried, and transferred to plastic scintillation
vials. BetaMax liquid scintillant (5 mL) was added, the vials
were shaken, stored overnight in darkness (to dissipate chemiluminescence), and the radioactivity was determined by liquid
scintillation spectroscopy in a Beckman LS- lOOC instrument.
3H counting efficiency was approximately 30%. Counting
times were sufficient to achieve a counting error of not more
than 0.2%. Control incubations consisted of boiled enzyme
extracts incubated with complete assay mixture.
Isolation of [3H]Glutamyl-tRNA for Use as Enzyme
Substrate
For determination of the ability of extracts from light- and
dark-grown cells to form glutamyl-tRNA that can serve as a
substrate for ALA formation, the 3H-based glutamyl-tRNA
synthetase assay reaction, as described above, was terminated
by the addition of 2 mL of aminoacyl-tRNA buffer (100 mM
Mes, 100 mm glutamate, 10 mM MgCl2 [pH 5.8]) and the
mixture was shaken with 3 mL of phenol (preequilibrated
with aminoacyl-tRNA buffer). After separation of phases by
centrifugation, the aqueous phase was extracted with 3 mL of
chloroform:isoamyl alcohol (24:1, v/v). The glutamyl-tRNA
was precipitated by the addition of 300 uL of 20% (w/v)
aqueous Na-acetate and 8.25 mL (2.5 volumes) of absolute
ethanol, and holding at -75°C for 1 h. The precipitate was
collected by centrifugation, washed with ice-cold 70% ethanol,
dried on ice with air, and dissolved in 200 ,uL of RNA buffer
(25 mm Mes, 10 mM glutamate, 10 mM MgC12 [pH 5.8]). The
final tRNA concentration was approximately 125 A260 units
per mL.
For preparation of glutamyl-tRNA for use as substrate in
the assay of reductase activity, tRNA extracted from wildtype cells grown in the light (250 A260 units) was precipitated
with 2.5 volumes of absolute ethanol at -75°C for 1 h in the
reaction tube. The precipitate was pelleted by centrifugation
at 13,000g for 30 min. The reaction mixture (1.0 mL) contained the pelleted tRNA, 5 mm ATP, 100 gM glutamate
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1368
MAYER AND BEALE
having 3 x 108 cpm of [3,4-3H]glutamate (the final specific
radioactivity was 3000 cpm pmol-'), and extract from lightgrown cells (1.9 mg of protein). The reaction was initiated by
addition of glutamate, allowed to proceed for 30 min at 30°C,
and terminated by addition of 2 mL of aminoacyl-tRNA
buffer. Immediately before addition of the termination buffer,
5 ML of incubation mixture was removed and transferred to
a glass fiber filter for determination of glutamyl-tRNA product. The reaction mixture and the filter were processed as
described above. The dried glutamyl-tRNA was dissolved in
half-strength aminoacyl-tRNA buffer at a concentration of
500 A260 units per mL, and aliquots were stored at -75°C.
Plant Physiol. Vol. 94,1990
was column buffer containing 5 mM levulinic acid, 40 pAM
GSA, and extract from cells grown in the light or the dark
(1.5 mg protein). Incubations were carried out at 30°C for 20
min in 300 ,L reaction volume. Reactions were initiated by
Addition of substrate, and ALA was isolated and quantitated
spectrophotometrically as described above. In some cases,
control reactions to correct for nonenzymatic conversion of
GSA to ALA were carried out with enzyme that was denatured
by heating to 1 00°C for 5 min, cooled, and then incubated
with the reaction mixture. In other cases, the control reaction
contained enzyme that was inactivated by preincubation for
20 min at 30°C with 5 ,uM AHA (11), a potent irreversible
suicide inactivator of GSA aminotransferase (2).
In Vitro Conversion of [3H]G1utamy1-tRNA to ALA
Incubations were carried out for 60 min at 30°C, in 250 yL
of column buffer containing 5 mm levulinate, 2.5 mm Mes,
1.0 mm NADPH, 1.0 mm unlabeled glutamate, enzyme extract from light-grown cells (630 ug of protein), and 2.5 A260
units of [3H]glutamyl-tRNA that was precharged by the enzyme being tested and containing 1.3 to 2.0 x 105 cpm of 3H
(the specific radioactivity of the glutamate in [3H]glutamyltRNA was 3000 cpm pmol-'). Before the reactions were
started by the addition of charged RNA to the mixtures, 5 ,uL
of each tRNA was applied onto a filter, and a filter binding
assay was performed to determine the initial concentration of
[3H]glutamyl-tRNA substrate. ALA was isolated as described
above and the ALA pyrrole was extracted into ethyl ether as
previously described (1). The combined ether phases containing the pyrrole were evaporated to dryness in plastic scintillation vials, shaken with 5 mL of BetaMax scintillation cocktail, and the radioactivity determined by liquid scintillation
spectroscopy.
Glutamyl-tRNA Reductase Assay
Reductase activity was quantitated by measuring the ability
of varying amounts of an enzyme extract to form ALA in the
presence of constant levels of both precharged [3H]glutamyltRNA and GSA transaminase. Constant GSA aminotransferase activity was achieved by supplementing the incubations
with adjusted amounts of BU obtained from light-grown cells
by fractionation as described above. This fraction contains
aminotransferase activity, but not reductase (40). The assay
was a slight modification of the procedure to assay in vitro
conversion of [3H]glutamyl-tRNA to ALA. Incubations were
carried out at 30°C for 5 min in 250 ,uL of column buffer
containing 5 mM levulinic acid, 1.0 mm NADPH, 2.5 A260
units of [3H]glutamyl-tRNA having 3.6 x 106 cpm (the specific radioactivity of the glutamate in [3H]glutamyl-tRNA was
3000 cpm pmol-'), and the enzyme extract being tested. ALA
isolation, extraction, and liquid scintillation spectroscopy
were performed as described above.
GSA Aminotransferase Assay
Aminotransferase activity was measured by assaying conversion of chemically synthesized GSA to ALA by cell extracts. GSA was prepared from 4-amino-5-hexenoic acid (vinyl GABA) by the method of Gough et al. (15). Assay medium
Other Procedures
Cell population densities were determined with a Coulter
Counter (model ZBI, Coulter Electronics). Protein concentrations were determined by the dye-binding method of Bradford
(7) using BSA as the standard. Chl was determined by measurement of A665 in methanol extracts of cells, and using an
absorption coefficient of 6.66 x 104 L mol-' cm-' for Chl a,
derived from the data in MacKinney (22). Pancreatic RNase
A (Sigma type I-AS) was dissolved in RNase buffer (1.0 M
Tris-HCl, 15 mm NaCl [pH 7.5]), heated for 8 min at 100°C,
cooled slowly, and stored at -20°C (23). Glutamyl-tRNA
synthetase assays and preparation of [3H]glutamyl-tRNA were
performed in Corex tubes siliconized by rinsing with Sigmacote prior to autoclaving.
Chemicals
L-[3,4-3H]Glutamate and L-[ 1-'4C]glutamate were purchased from Du Pont-New England Nuclear. AHA and 4amino-5-hexanoic acid were generous gifts from Merrell Dow
Research Institute, Cincinnati, OH. Wratten filters were purchased from Eastman Kodak Co. DEAE-cellulose DE-23 was
from Whatman. Glass fiber filter discs were from Schleicher
& Schuell. BetaMax liquid scintillation cocktail was from ICN
Biomedicals. Miracloth was from Calbiochem-Behring.
All other chemicals were from Sigma, Fisher, and Research
Organics.
RESULTS
Effects of Various Light Intensities on Chi Content and in
Vitro ALA-Forming Activity
Cells that had been growing in complete darkness for many
generations were transferred to light of different incident
intensities and allowed to grow for 48 h before harvesting. In
this experiment, the standard cool-white plus red fluorescent
light source was the same for all cultures, and the intensity
was adjusted by placing layers of Miracloth between the light
source and the culture flask. Chl was measured at the time of
harvest. For determination of ALA-forming activity, 1.0-mL
incubations contained 2.6 to 3.7 mg of protein (prepared from
cell extracts with the differential (NH4)2SO4 precipitation step
omitted) and 10 A260 units of RNA derived from light-grown
cells.
Cells kept in the dark did not synthesize Chl, and the very
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1369
LIGHT REGULATION OF ALA BIOSYNTHETIC STEPS IN EUGLENA
1000
2.00
.a)
1.50
QL
E
c)
z
1.00
E
c
c
E
0
0.50
0
0.00
0
5
10
15
20
25
30
the light meter was equipped with a pyranometer probe having
red response extending to 900 nm. Light intensity was
measured after serially passing through the blue filter, the red
filter (to remove blue light), and an interference filter having
a 10 nm passband centered at 720 nm. The intensity at 715
to 725 nm, after correction for passband absorbance of the
red and interference filters and conversion of the pyranometer
reading to photon flux, was determined to be 0.17 ,E m-2
s-'. To determine whether the far-red light passing through
the blue filter was perceived by the cells, a culture of darkgrown cells was transferred to light that was passed serially
through the red and blue filters before illuminating the culture. After 48 h of growth under this illumination, the Chl
content was 6 nmol g-' cells and the in vitro ALA formation
activity was 0.41 nmol g-' protein, compared to the respective
values of 6 nmol Chl g-' cells and 0.14 nmol ALA mg-'
protein for cells kept in the dark.
a
Ez
-
35
Light Intensity (1±E m-2 s-1)
Figure 2. Chl content and in vitro ALA-forming activity of dark-grown
cells transferred to light of various intensities.
small amount present in the culture that remained in the dark
probably represents the Pchlide that was present immediately
before harvest (Fig. 2). Significant amounts of Chl were synthesized even at 1 gE m-2 s-', the lowest tested light intensity.
Light at or above 8 ,uE m-2 s-' intensity was approximately
equally effective in inducing Chl synthesis.
In agreement with previously reported results (24), extracts
of cells that were grown in the dark for long periods had very
little ALA-forming activity (Fig. 2). Light at or above 8 ,uE
m-2 s-' intensity was approximately equally effective in inducing in vitro ALA-forming activity. After 48 h in the light,
both the Chl content and the in vitro rate of ALA formation
reached approximately half the levels in cells grown continuously in the light (1670 nmol Chl g-' cells and 3.92 nmol
ALA mg-' protein, respectively).
Effects of Growth in White, Red, and Blue Light on in
Vitro ALA-Forming Activity
Cells that had been growing in complete darkness for many
generations were transferred to white or colored light and
allowed to continue growing for 48 h. All incident light
intensities were equalized to 13 ,E m-2 s-' by combining
layers of Miracloth with the colored filters and/or by supplementing the standard mixture of cool-white and red fluorescent lights with incandescent light. ALA formation was measured in 1-mL incubations containing 5 mg of protein (prepared from cell extracts with the differential (NH4)2SO4
precipitation step omitted) and 10 A260 units of RNA derived
from light-grown cells.
Blue light was more effective than red light in inducing
ALA-forming activity, but neither blue light (400-480 nm)
nor red light (650-700 nm) alone induced as much activity
as white light (Table I). However, a combination of blue plus
red light was as effective as white light.
Because the blue filter also transmits light above 720 nm,
it was necessary to estimate the amount of light in this spectral
region that was incident on the cultures and to determine
whether the cells respond to this light. To measure the incident
intensity of the blue filtered light in the 715 to 725 nm region,
Effect of Growth in Light or Dark on Extractable
Glutamate-Accepting tRNA Levels
Cells grown in the light or dark for many generations were
harvested and the extracted tRNA was tested for the ability
to become charged with glutamate. ['4C]Glutamyl-tRNA formation was assayed by incubating extract of light-grown cells
with ['4C]glutamate plus tRNA isolated from light- or darkgrown cells. Incubations contained 5 A260 units of tRNA and
1.03 mg of extracted protein from light-grown cells.
Equal amounts of tRNA from light and dark-grown cells
increased glutamyl-tRNA formation by 280 and 367%, respectively, over the level supported by endogenous tRNA
present in the enzyme extract that was obtained from lightgrown cells (Table II). The somewhat greater amount of
glutamyl-tRNA formed per A260 unit of added tRNA derived
from dark-grown cells, compared to that of light-grown cells,
suggests that tRNA from dark-grown cells has a greater fractional abundance of tRNAG"U than tRNA derived from lightgrown cells.
Table I. In Vitro ALA-Forming Activity of Dark-Grown Cells
Transferred to Light of Various Wavelength Ranges
Dark-grown cells were kept in the dark or transferred to light
having the indicated wavelength range, and allowed to continue
growing heterotrophically for 48 h before harvesting. All light intensities were equalized to a total fluence rate of 13 pE m-2 s-1. For
determination of ALA-forming activity, cell extracts were incubated
for 60 min at 300C in 1.0 mL of assay buffer containing 5 mg of
protein and 10 A260 units of RNA derived from light-grown cells.
Light
Wavelength Range
nm
White
Red
Blue
Red + blue
Dark control
400-700
650-700
400-480
400-480 + 650-700
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ALA Formation
nmol mg' protein
%
2.62
1.44
2.21
2.61
0.14
100
55
84
100
5
1370
Plant Physiol. Vol. 94, 1990
MAYER AND BEALE
Table II. Glutamate-Accepting Ability of tRNA Extracted from Lightand Dark-Grown Cells
Glutamyl-tRNA Formation
Source of Added tRNA
net increase
cpm
Endogenous tRNA only
Light-grown cells
Dark-grown cells
None, not incubated
2,340
8,220
10,190
200
0
280
367
Effect of Growth in Light or Dark on Extractable
Glutamyl-tRNA Synthetase Activity
Cells that were grown for many generations in the light or
dark were harvested and the enzyme extracts were tested for
the ability to charge tRNA with glutamate. [3H]GlutamyltRNA formation was assayed by incubating extract of lightor dark-grown cells containing 0.63 mg of protein, with [3H]
glutamate plus tRNA isolated from light-grown cells.
Both cell extracts were able to charge the tRNA, but the
extract from dark-grown cells was only 65% as active as the
extract from light-grown cells (Table IV). Heat-denatured
enzyme was inactive.
Table Ill. Ability of tRNA from Light- and Dark-Grown Cells to
Support ALA Formation with Enzyme Extract from Light-Grown Cells
Light-grown cells
Dark-grown cells
None
ALA Formation
1.07
0.07
Formation
x
cpm 10-6
Light-grown
Light-grown, heat-denatured
Dark-grown
Dark-grown, heat-denatured
tRNA extracted from cells grown in the light or dark for
many generations was tested for the ability to support in vitro
ALA formation when added to enzyme extract of light-grown
cells.
Cell extract was preincubated with 75 ng of RNase for 20
min at 30°C in 250 ,uL of column buffer containing 1.2 mg
of protein extracted from light-grown cells. Next, RNasin
(200 units) was added and incubation continued for 5 min.
Then, 5 A260 units of tRNA extracted from light- or darkgrown cells was added along with substrate and cofactors and
incubation continued for 60 min.
The RNase digestion was effective in removing most of the
endogenous tRNA capable of supporting ALA formation, and
the background level of activity was only 2% of the activity
in extract that was supplemented with tRNA from light-grown
cells. tRNA from light-grown cells was approximately three
times more effective than tRNA from dark-grown cells in
supporting ALA formation (Table III).
nmol mg-1 protein
3.10
Glutamyl-tRNA
Cell Extract
(%/)
Effect of Growth in Light or Dark on Extractable tRNA
That Is Capable of Supporting in Vitro ALA Formation
from Glutamate
Source of Added tRNA
Table IV. Glutamyl-tRNA Synthetase Activity in Enzyme Extracts
from Light- and Dark-Grown Cells
%
100
35
2
1.580
0.005
1.020
0.001
%
100
0
65
0
Effect of Growth in Light or Dark on Extractable Activity
Capable of Forming Glutamyl-tRNA That Can
Subsequently Be Converted to ALA
The [3H]glutamyl-tRNA produced in the above experiment
was isolated and used as a substrate for conversion to ALA
by extract of light-grown cells. Incubation conditions in the
ALA-formation assay were chosen so that free [3H]glutamate,
which might arise by hydrolysis of [3H]glutamyl-tRNA, could
not be effectively converted to ALA. The [3H]glutamate control sample contained, instead of precharged [3H]glutamyltRNA, 2.5 A260 units of uncharged tRNA derived from lightgrown cells plus the indicated amount of [3,4-3H]glutamate.
Glutamyl-tRNA formed by extracts of both light- and darkgrown cells was comparably effective as a substrate for ALA
formation. During the reaction, between 34 and 44% of the
substrate glutamyl-tRNA was converted to ALA (Table V).
Effect of Growth in Light or Dark on Extractable GSA
Aminotransferase Activity
Cells grown in the light or dark for many generations were
harvested and the enzyme extracts tested for activity in converting chemically synthesized GSA to ALA. There is a considerable rate of nonenzymatic conversion of GSA to ALA,
and this rate is influenced by the presence of proteins and
other cellular components. Therefore, two types of control
incubations were carried out: with heat-denatured enzyme
and with enzyme that was inactivated by preincubation with
AHA, an irreversible suicide substrate analog for GSA aminotransferase (2).
In experiment I, 250-AL incubations contained 0.6 mg of
cell protein and control samples had enzyme extract that was
heat denatured prior to its addition to the incubation. In
experiment II, 300 uL incubations contained 1.45 mg of cell
protein (prepared from cell extract with the differential
(NH4)2SO4 precipitation step omitted) and control samples
Table V. Formation of Glutamyl-tRNA by Enzyme Extracts from
Light- and Dark-Grown Cells that is Subsequently Convertible to
ALA by Enzyme Extract from Light-Grown Cells
Cell Extract for
Charging Reaction
Light-grown
Dark-grown
[3H]Glutamate control
[3H]Glutamyl-tRNA Added
to ALA-Forming Reaction
ALA Formation
cpm x 10-3
197
127
300
cpm x 10-3% conversion
34
67.3
44
54.6
0
0.1
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LIGHT REGULATION OF ALA BIOSYNTHETIC STEPS IN EUGLENA
Table VI. Aminotransferase Activity in Enzyme Extracts from Lightand Dark-Grown Cells
Cell Extract
Experiment I
Light grown
Light-grown, heat-denatured
Dark-grown
Dark-grown, heat-denatured
Experiment II
Light-grown
Light-grown, AHA preincubated
Dark-grown
Dark-grown, AHA preincubated
A553
Net ALA Formation
nmol mg-' protein
0.222
0.076
0.071
2.14
0.00
0.088
0.258
0.046
0.060
0.054
1.93
0.05
were preincubated with 5 ,uM AHA for 20 min before the
addition of GSA. Values for nonenzymatic ALA formation,
measured in control reactions, were subtracted from the experimental values to yield net ALA formation.
AHA and heat denaturation appeared to inhibit total ALA
formation by comparable amounts, and the residual nonenzymatically formed ALA values were subtracted from the
experimental values to yield net aminotransferase activity
(Table VI). The calculated net aminotransferase activity in
dark-grown cells was near the limit of detectability, and may
not differ significantly from zero.
Effect of Growth in Light or Dark on Extractable
Glutamyl-tRNA Reductase Activity
Reductase activity is measured in a coupled enzyme assay
in which the presumed product, GSA, is converted to ALA
by aminotransferase. Extract from dark-grown cells has little
or no aminotransferase activity. Therefore, to determine the
reductase activity in this extract, it was necessary to supplement it with Blue-Sepharose fraction BU from extract of lightgrown cells, which contains aminotransferase but is largely
depleted of reductase activity (40). In initial experiments
comparing the reductase activity in extracts of light- and darkgrown cells, a quantity of fraction BU enzyme was added to
extract of dark-grown cells to bring the aminotransferase
activity to the level present in extract from an equivalent
quantity of light-grown cells. Under these conditions, there
appeared to be equal reductase activity in the extracts of lightand dark-grown cells (data not shown).
However, it was realized that, in these incubations, if the
aminotransferase were the rate-limiting activity, then differences in reductase activity might not be measurable. Therefore, a series of incubations was carried out, in which the
amount of total aminotransferase activity was held constant,
while the amount of cell extract that was being tested for
reductase activity was progressively lowered. In this incubation series, the amount of aminotransferase activity used was
equal to the activity present in extract from 100 mg of lightgrown cells. The amount of extract that was tested for reductase activity ranged from the equivalent of 2 to 21 mg of
extracted cells.
Incubation was for 5 min with protein derived from lightor dark-grown cells. To allow temperature equilibration to
1371
occur, incubation was preceded by a 3-min preincubation of
all ingredients except the [3H]glutamyl-tRNA. The [3H]glutamyl-tRNA was prepared in a separate incubation and subsequently isolated from a charging reaction containing lightgrown cell extract, [3,4-3H]glutamate, and tRNA derived from
light-grown cells.
At the lowest ratio of cell extract to aminotransferase in
this incubation series, extract from light-grown cells stimulated ALA formation over four times as much as did extract
from dark-grown cells. As the ratio of cell extract to aminotransferase in the incubations was progressively raised, differences in the increased ALA formation in incubations containing extracts from light- and dark-grown cells became progressively less (Fig. 3). At the highest tested ratio, the incubation
containing extract from light-grown cells had about twice the
increased ALA formation as those containing dark-grown cell
extract. ALA that was formed in incubations containing fraction BU only is attributed to residual reductase activity in
fraction BU.
Activity of Reductase from Dark-Grown Cells in the
Absence of Aminotransferase Activity
In the experiments described above, reductase activity was
measured in a coupled enzyme assay in which the presumed
product, GSA, was converted to ALA by aminotransferase. It
was important to determine whether activity of the reductase
enzyme depends on the simultaneous activity of aminotransferase.
Extract from dark-grown cells, which contains little or no
aminotransferase activity, was incubated with [3H]glutamyltRNA to generate GSA. This incubation was for 10 min at
30°C in 250 ,uL of pH 7.9 buffer containing 1.0 M glycerol,
100 mM Tricine, 15 mM MgCl2, 10 mM Mes, 10 mm unlabeled
glutamate, 5 mM levulinate, 1.0 mM DTT, 1.0 mm NADPH,
20 ,uM PALP, 25 A260 units of [3H]glutamyl-tRNA having
2.37 x 106 cpm (the specific radioactivity of the glutamate in
[3H]glutamyl-tRNA was 3000 cpm pmol-'), and 0.62 mg of
12,000
10,000
8,000
E
0
6,000
-J
co)
4,000
2,000
0
-
0.00
0.02
0.04 0.06 0.08
Protein (mg)
0.10
0.12
Figure 3. Glutamyl-tRNA reductase activity in enzyme extracts from
light- (O) and dark-grown (*) cells.
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Copyright © 1990 American Society of Plant Biologists. All rights reserved.
1372
MAYER AND BEALE
Table VIl. Activity of Reductase in the Absence of Aminotransferase
Sample
ALA Formation
Experimental
Fraction BU pretreated with 5 AM AHA
[3H]Glutamate only, no cell extract in incubations
RNase treatment before first incubation
195,500
37,600
3,000
2,100
cpm
protein from extract of dark-grown cells. After this incubation,
the remaining glutamyl-tRNA was destroyed by the adding
25 units of RNase and continuing the incubation for 5 min.
Then, 0.53 mg of Blue-Sepharose fraction BU of cell extract
from light-grown cells, which contains aminotransferase, was
added and the incubation continued for 20 min. The formation of [3H]ALA, produced from the [3H]GSA that was
formed during the first incubation, was measured. As a control, an identical experiment was carried out, using fraction
BU that had been pretreated with AHA to inactivate aminotransferase.
Extract from dark-grown cells was able, during the first
incubation, to convert [3H]glutamyl-tRNA into a product
that was subsequently transformed to ALA in the second
incubation, in the absence of [3H]glutamyl-tRNA, by extract
of light-grown cells (Table VII). More than 8% of the label in
the [3H]glutamyl-tRNA was transferred to ALA. Pretreatment
of fraction BU with AHA inhibited the formation of ALA by
more than 80%.
Relative in Vitro Rates of the Three Enzymes and Overall
Conversion of Glutamate to ALA in Extracts of Light- and
Dark-Grown Cells
Data from Tables I, V, and VII, and Figure 3, were recalculated to express enzyme activity and rate of ALA formation
in units of nanomoles of product formed per h per g fresh
weight of extracted cells (Table VIII). The values for reductase
activity were taken from the points on Figure 3 representing
the lowest tested quantity of cell extract, because these had
the highest specific activity and showed the greatest difference
between dark- and light-grown cell extracts. The values for
aminotransferase activity were taken from experiment II of
Table VI, and the value for the dark-grown cell extract should
be considered to be the maximum possible value.
Because Chl is the predominant tetrapyrrole end product
in light-grown cells, its concentration can be used to estimate
the in vivo rate of ALA formation, based on the fact that one
Plant Physiol. Vol. 94,1990
Chl molecule is formed from eight ALA molecules. The
calculated in vivo rate of ALA formation in cells grown
continuously in the light, using a cell population doubling
time of 12 h and a Chl content of 1670 nmol g-' cells, is 800
nmol h-' g-I cells.
Because the calculated rate of in vivo ALA synthesis is
much higher than any of the recovered in vitro enzyme
activities, it is not possible to directly relate these in vitro rates
to in vivo enzyme rates. Nevertheless, it is of interest that the
aminotransferase is the only enzyme whose in vitro light/dark
activity ratio closely parallels that of both the in vitro rate of
ALA formation from glutamate and the calculated in vivo
rate of Chl synthesis.
DISCUSSION
Euglena is unique among organisms in that it has been
shown to form ALA by both the tRNA-dependent five-carbon
pathway and by the ALA synthase-catalyzed condensation of
glycine with succinyl-CoA. In vivo, the five-carbon pathway
is the sole source of Chl precursors in Euglena, and the
pathway is inactive in dark-grown cells (36). ALA synthase,
on the other hand, is the sole source of mitochondrial tetrapyrrole precursors in Euglena, and may also supply other
cellular tetrapyrrole end products in the dark (36). In vitro,
ALA synthase activity is much higher in extracts of darkgrown cells than in light-grown cell extracts, and the extractable activity declines precipitously within the first few hours
after transfer of dark-grown cells to the light (13). In this
report we have determined that growth in the dark or light
results in differences in the extractable activities of the three
known enzymes of the five-carbon pathway, as well as the
relative levels of the tRNAG"U that is required for ALA formation from glutamate.
Cells that were adapted to the dark by being cultured for
many generations in the dark have very low in vitro ALAforming activity. At 48 h after transfer to light of 8 ,uE m-2
s- or greater incident intensity, in vitro ALA-forming activity
rises to approximately half the level present in extracts of cells
grown in the light for many generations.
The comparable effectiveness of red and blue light indicates
that either the red/blue photoregulatory system or the blue
system alone can induce the five-carbon pathway. Blue light
is nearly as effective as white light, while red light alone is less
effective than blue or white light. Full induction equal to that
produced by white light is not reached by either red or blue
light alone, and requires light of both spectral regions. It is of
interest that the same pattern in relative effectiveness of white,
Table Vil. Comparison of Glutamyl-tRNA Synthetase, Glutamyl-tRNA Reductase, GSA
Aminotransferase Activities, and Overall Conversion of Glutamate to ALA in Extracts from Light- and
Dark-Grown Cells
Cell Extract
Glutamyl-tRNA
Light-grown
Dark-grown
Ratio light-/dark-grown
29.6
16.9
1.8
Synthetase
Glutamyl-tRNA
GSA
Reductase
Aminotransferase
nmol product h 1 g 1 fresh wt cells
7.6
1.8
4.2
ALA
Formation
92.6
2.0
39.8
2.1
46.3
19.0
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LIGHT REGULATION OF ALA BIOSYNTHETIC STEPS IN EUGLENA
blue, and red light was found in the elimination of the lag
phase of Chl synthesis by preillumination of dark-grown cells
(17).
To measure the relative levels of the relevant tRNAGIU in
light- and dark-grown cells, the tRNA was tested for the ability
to support ALA synthesis in extract from light-grown cells.
This was necessary because there may be several tRNAs that
do not function in ALA synthesis but are nevertheless capable
of being charged with glutamate, including nonplastid
tRNAGIUs and possibly tRNAG'InS (32). Although total glutamate-accepting ability in extracts of dark- and light-grown
cells is comparable, the amount of the relevant tRNAG"U that
can support ALA synthesis extracted from light-grown cells is
three times higher than the amount extracted from darkgrown cells.
To measure the cellular levels of the relevant glutamyltRNA synthetase, the relative ability of extracts from darkand light-grown cells to form glutamyl-tRNA that could subsequently be used as a substrate for ALA synthesis was determined. This was necessary because some fraction of the total
cellular glutamyl-tRNA synthetase (e.g. cytoplasmic or mitochondrial enzyme) may be unable to charge the tRNAGIU that
participates in ALA formation. Total glutamyl-tRNA synthetase activity, measured during a short, 5-min charging incubation, is substantially lower in extracts of dark-grown cells
compared to the activity in light-grown cell extracts. However,
the [3H]glutamyl-tRNA formed by extracts of light- and darkgrown cells during the short incubation is comparably effective, per unit of total [3H]glutamyl-tRNA administered, as a
substrate for conversion to ALA after isolation and subsequent incubation with extract from light-grown cells. Moreover, the fraction of total cellular tRNAGIU that participates
in ALA formation is chargeable to approximately the same
level by the enzyme from dark- and light-grown cells, in a
longer, 60-min incubation. These results indicate that lightand dark-grown cells contain glutamyl-tRNA synthetase that
is equally active in charging the tRNA that participates in
ALA formation.
Reductase activity is over four times higher in extract of
light-grown cells than in dark-grown cell extract, when measured in the coupled assay with the ratio of reductase to
aminotransferase adjusted to maximize the effect of reductase
activity on the overall reaction.
Aminotransferase activity shows the greatest difference between dark- and light-grown cells. Activity is undetectable or
barely above the background in extract of dark-grown cells,
and is at least 40-fold higher in light-grown cell extract.
It is of interest that the levels of both the tRNA and enzymes
that function in ALA synthesis via the five-carbon pathway
are lower in extracts of dark-grown cells than in light-grown
cell extracts. In vitro ALA-formation from glutamate has been
reported to be lower in extracts of dark-grown Chlorella cells
(37) and plastids of etiolated maize (16) and barley (19) leaves,
and cucumber cotyledons (35). Effects of light or dark during
growth or germination on in vitro activities of specific enzymatic steps in ALA synthesis have been reported only for the
aminotransferase of barley plastids. Plastid extracts from
greening tissues are considerably more active than those of
etioplasts or mature green leaves ( 18). In barley leaves, gabaculine-induced accumulation of GSA was reported to be
1373
stimulated by light (20). This suggests that both the aminotransferase and some step prior to the aminotransferase are
regulated by light in barley, but the in vivo mode of regulation
of the earlier step is not known. The Euglena results for
tRNAGIU are in contrast to those reported for other systems.
Etiolated barley plastids contain as much tRNAGIu as plastids
isolated from greening leaves (6). Similarly, in a Chlorella
strain that requires light for greening, the tRNA that supports
ALA synthesis does not vary with light or dark growth conditions (39). It was reported earlier that some plastid tRNAs,
including tRNAG"u (3) and plastid aminoacyl-tRNA synthetases (26) are induced by light in Euglena. Effects of light on
the level of glutamyl-tRNA synthetase was not reported in
these studies.
The fact that the levels of more components of the ALAforming system are effected by light versus dark growth in
Euglena than has been reported for plants and other algae
may be related to the fact that Euglena cells that are grown
in the dark contain proplastids (29), whereas in the other
species etioplasts are formed in the dark. Etioplasts may
represent a developmental alternative to the normal progression from proplastids to greening chloroplasts, and may be
poised for maximum rate of development of photosynthetic
competence upon light exposure following prolonged periods
in the dark. We propose that some macromolecular components of the ALA-forming system may accumulate in etioplasts to allow a more rapid rate of Chl synthesis after transfer
to the light than would be the case if all components had to
be synthesized de novo after light exposure.
None of the recovered in vitro enzyme activities approached
the in vivo rate of ALA synthesis, calculated from Chl content
and the rate of cell growth. This discrepancy prevents determination of the rate-limiting step in vivo by direct extrapolation from the in vitro rates of the enzymes. However, because
light- and dark-grown cells grew at similar rates and were
extracted identically, it is possible to compare each enzyme
and the overall rate of in vitro ALA formation with respect to
the activity ratio in extracts of light- and dark-grown cells. As
proposed above, the two- to four-fold differences in levels of
tRNAGlU, glutamyl-tRNA synthetase, and glutamyl-tRNA reductase in extracts of light- versus dark-grown cells may be
related to the state of plastid development and may have some
influence on the in vivo potential for ALA synthesis. However,
only aminotransferase activity differs sufficiently in extracts
of light- and dark-grown cells to correspond to both the
difference in the overall in vitro rate of ALA formation and
the difference in the in vivo rate of Chl synthesis. Two conclusions can be drawn from these results. First, the rate of ALA
formation in extracts of dark-grown cells is limited by the low
aminotransferase activity. Second, a deficiency in aminotransferase activity limits ALA synthesis via the five-carbon pathway in dark-grown Euglena cells. The second conclusion is
bolstered by the earlier observation that if aminotransferase
is inhibited in light-grown Euglena cells by administration of
gabaculine, Chl synthesis is completely inhibited, while heterotrophic cell growth continues (9).
In summary, light induces both the tRNAGIU and enzymes
that are components of the five-carbon ALA-forming pathway
in Euglena. Maximum induction requires both red and blue
light. Aminotransferase activity is the component most pro-
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1 374
MAYER AND BEALE
foundly affected by light. Aminotransferase activity may be
totally absent in dark-grown cells, and the absence may be
responsible for the very low level or absence of ALA synthesis
via the five-carbon pathway in the dark.
ACKNOWLEDGMENTS
We thank R. Solotaroff for performing the protein determinations, J. Biggins for providing the pyranometer and
interference filter, S. Rieble for helpful discussions, and A.
W. Holowinsky for critically reading the manuscript.
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Copyright © 1990 American Society of Plant Biologists. All rights reserved.