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Plant Physiol. (1998) 116: 1239-1248
Induced
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Under stress conditions such as
high light intensity or nutrient starvation, cells of the unicellular
alga Dunaliella bardawil overproduce 
-carotene, which
is accumulated in the plastids in newly formed triacylglycerol
droplets. We report here that the formation of these sequestering
structures and -carotene are interdependent. When the synthesis of
triacylglycerol is blocked, the overproduction of -carotene is also
inhibited. During overproduction of -carotene no up-regulation of
phytoene synthase or phytoene desaturase is observed on the
transcriptional or translational level, whereas at the same time
acetyl-CoA carboxylase, the key regulatory enzyme of acyl lipid
biosynthesis, is increased, at least in its enzymatic activity. We
conclude that under normal conditions the carotenogenic pathway is not
maximally active and may be appreciably stimulated in the presence of
sequestering structures, creating a plastid-localized sink for the end
product of the carotenoid biosynthetic pathway.
Carotenoids are vitally important in all photosynthetic membranes
because of their ability to prevent photooxidative damage and to
harvest light (for a recent review, see Frank and Cogdell, 1996 To investigate the apparently strict interdependence of carotenoid
overproduction and the formation of sequestering structures, we
exploited the unicellular alga Dunaliella bardawil as a
laboratory system inducible for enhanced carotenoid formation. When
exposed to stress conditions such as high light intensity or nutrient starvation, two stereoisomers of With this system we wished to address the causality question, i.e.
whether Growth and Induction Conditions
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
).
Underscoring their importance, chlorophylls and carotenoids are
synthesized in a quantitatively and qualitatively coordinated manner in
chloroplasts. Whenever this balance is strongly changed in favor of
carotenoids, the plastid ultrastructure is also changed and,
concomitantly, chlorophylls are degraded. The resulting chromoplasts are photosynthetically inactive, yellow to red in color, and
differentiate in specialized plant organs such as petals, roots, and
fruits. Chromoplasts are morphologically characterized by the absence of thylakoids and by the presence of newly formed structures in which
the overproduced carotenoids are sequestered (for a recent review of
chromoplast development, see Camara et al., 1995). These carotenoid-bearing structures may be plastoglobules (lipid droplets in
most carotenoid-bearing flower petals), crystals (e.g. in
Lycopersicon esculentum fruits), fibrils/tubules (e.g. in
Capsicum annuum fruits), or membranes (e.g. in
Narcissus pseudonarcissus petals). These structures probably
prevent products from overloading the chromoplast membranes, the site
of carotenoid formation.
-carotene, all-trans and
9-cis -carotene, accumulated, reaching up to 10% of the
cell's dry weight, with the pigment being deposited into plastid lipid
globules as the sequestering structure (Ben Amotz et al., 1982, 1988;
Ben Amotz and Avron, 1983; Jeminez and Pick, 1994). These lipid
structures are stabilized and maintained in size by a peripherally
associated 38-kD protein (Carotene globule
protein, Cpg), its formation induced in parallel with
-carotene accumulation (Katz et al., 1995). Although these
carotene-rich algal plastids still possess thylakoids and perform
photosynthetic reactions (Vorst et al., 1994) and thus do not represent
true chromoplasts, the underlying structural basis for carotene
accumulation is well preserved. Therefore, we considered D. bardawil to be a suitable model system for investigating the
above-mentioned interdependence.
-carotene accumulation is a consequence of enhanced lipid
production. Providing a sequestering structure could, for instance,
remove the -carotene end product from the biosynthetic machinery in
the membrane, leading to enhanced reaction velocities and thus
contributing to enhanced carotene formation. The results of the
molecular analyses presented here substantiate the validity of such a
sequestering hypothesis.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
). The algae were grown at 25°C in 0.5-L Erlenmeyer flasks containing 200 mL of medium under continuous shaking
and illumination (46 µmol m
2
s1, 400-700 nm). For induction of -carotene
synthesis, cells can be grown either in a sulfate-free medium
(MgCl2 instead of MgSO4) or
by applying high light intensities (690 µmol
m2 s1). High-light
induction led to a more rapid induction compared with sulfate
starvation and was therefore used in our experiments here. Cells were
harvested by centrifugation at 500g for 10 min and either
frozen at 
70°C or used directly.
Inhibition of Fatty Acid Biosynthesis in Vivo
For inhibiting the biosynthesis of acyl lipids during the induction of-carotene synthesis, we used the herbicide sethoxydim (2-[1-{ethoxyimino}butyl]-5-[2-{ethylthio}propyl]-3-hydroxy-2-cyclohexen-1-one; Riedel-de Haen, Seelze, Germany), which was added to cells at the
onset of induction at concentrations ranging from 10 to 50 µm. Alternatively, the antibiotic cerulenin
(2,3-epoxy-4-oxo-7,10-dodecadienamide; Sigma) was used at final
concentrations of 0.5 to 8 µm.
Incubation Assays and Analysis of Reaction Products
Lipids were radioactively labeled in vivo by adding 138.73 kBq NaH14CO3 (20.4 MBq mmol1, Amersham) to cultures at the onset of
high-light treatment. After 48 h the cells were pelleted
(500g, 10 min) and lipids were extracted by the addition of
2 mL of chloroform:methanol (2:1, v/v). The lipids were then separated
by two-dimensional TLC (first dimension, chloroform:methanol:water,
65:25:4 [v/v/v]; second dimension, chloroform:methanol:ammonium
hydroxide [25% solution]:isopropylamine, 65:35:5:0.5 [v/v/v/v])
and visualized by spraying the plates with rhodamine-tinopal (Kleinig
and Lempert, 1970). The identification of individual lipids was based
on co-chromatography with known standards (Kleinig and Liedvogel,
1978). The separation between chlorophylls and carotenoids was achieved
with a second system consisting of silica gel plates developed with the
solvent system hexane:diethylether:formic acid, 80:20:2 (v/v/v).
Aliquots of individual lipids scraped off from the plates were mixed
with 5 mL of scintillation fluid (Rotiszint-Ecoplus, Roth, Karlsruhe, Germany) and 0.2 mL of 1 n HCl to prevent the
phosphorescence caused by the rhodamine reagent.
1, Amersham). Incubation was carried out at
25°C for 2 h. The reaction was stopped by adding 2 mL of
chloroform:methanol, 2:1 (v/v). After the sample was mixed and phase
separated by brief centrifugation, the acetate incorporation into
lipids was quantified by liquid-scintillation counting.
1; Amersham). After incubation for 1 h
at 26°C, 1 mL of a solution containing 1 m KCl in 0.2 m phosphoric acid (Hajra, 1974) was added. Lipids were
extracted with 2 mL of chloroform:methanol, 2:1 (v/v), and separated on
silica gel plates (Kieselgel GF254, Merck,
Darmstadt, Germany) using petroleum ether:chloroform:acetone, 50:20:2
(v/v/v). Individual bands were scraped off and quantified by
liquid-scintillation counting, as above.
1; Amersham). The reaction products were
extracted with chloroform:methanol, 2:1 (v/v), and analyzed by HPLC as
described by Beyer and Kleinig (1992).
in 0.5 mL of
buffer containing additionally 0.5 mm ATP, 138.75 kBq
NaH14CO3 (20.4 MBq
mmol1, Amersham), and 0.3 mm
acetyl-CoA. The sample was preincubated at 30°C for 3 min and the
reaction started by adding acetyl-CoA. The incubation was carried out
at 30°C for 20 min. The reaction was stopped by adding 50 mL of 6 n HCl and vigorously mixing. Aliquots of 100 mL were dried
on 2- × 2-cm squares of Whatman 3M paper. The
radioactivity representing the incorporation into acid-stable products
was determined by scintillation counting. Inhibition experiments were
carried out using sethoxydim or cerulenin at 50 µm and 8 mm, respectively.
[35S]Met Labeling of Inhibited Cells
Uninhibited and (sethoxydim- or cerulenin-) inhibited cells were pelleted as above, washed twice with medium, and resuspended in Met assay medium (Difco, Detroit, MI). The cultures were labeled with 37 kBq [35S]Met (111,000 GBq mmol1; Amersham) for 1 h. The proteins
were precipitated (Chua, 1980) and separated by SDS-PAGE (see below).
The dried gel was then exposed to film for autoradiography. An aliquot
of each sample was quantified by scintillation counting.
Analytical Methods
Carotenes and Chlorophylls
Chlorophyll was assayed photometrically according to the method of Arnon (1949). -Carotene was quantified photometrically using as a
molar extinction coefficient 
450 nm = 134,500 L mol1 cm1.
Lipids and Fatty Acids
Fatty acid residues were estimated photometrically after saponification (1 mL of 10% KOH in ethanol for 2 h) according to the method described by Duncombe (1963). Individual fatty acids were
identified after methylation using diazomethane by GLC (Kleinig et al.,
1986).
Proteins
Soluble and membrane-bound proteins were precipitated with 10% TCA in 80% acetone (Chua, 1980) and quantified (Schacterle and
Pollack, 1973). SDS-PAGE was performed on 10 to 12% polyacrylamide gels according to the method of Laemmli (1970). For western analysis the proteins were electrotransferred semidry onto nitrocellulose membranes using a LKB-Multiphor apparatus (LKB/Pharmacia, Freiburg, Germany). Blots were examined by staining reversibly with 0.02% (w/v)
Ponceau S in 3% (v/v) TCA. For immunostaining and detection we used
the enhanced chemiluminescence detection system (Amersham) according to
the manufacturer's instructions. The primary antibodies anti-phytoene
synthase and anti-phytoene desaturase raised against the overexpressed
proteins (Al-Babili et al., 1996; Schledz et al., 1996), both from
Narcissus pseudonarcissus, were used.
Prenylphosphates
Prenylphosphates were converted to the corresponding alcohols by the addition of 50 mL of alkaline phosphatase (1400 units mL1) in 1 mL of lysed cells resuspended in
buffer using a closed-loop device (Grob and Zürcher, 1976). The
incubation took place for 12 h at room temperature with vigorous
mixing. The resulting products were analyzed by GLC (Mettal et al.,
1988).
DNA/RNA Techniques
Standard molecular biological techniques were carried out according to the method of Sambrook et al. (1989). RNA was isolated from frozen cells (100 mg) harvested at different stages of induction and isolated according to the method of Chirgwin et al. (1979). The
RT-PCR assay was performed in an incubation buffer containing 5 mm MgCl2, 50 mm KCl, 50 mm Tris-HCl, pH 8.3, 2 mm deoxyribonucleotide triphosphates, 10 mm DTT, 100 ng of the respective
downstream primer, and 20 units of Superscript II (GIBCO-BRL) at 42°C
for 30 min in a total volume of 10 mL. The reaction was stopped by heating the samples to 94°C for 5 min. The tubes were transferred to
ice and 40 mL of precooled PCR master mixture containing 50 mm KCl, 1.5 mm MgCl2, 10 mm Tris-HCl, pH 9.0, 100 ng of upstream primer, and 0.5 units of Taq polymerase (Pharmacia) was added. PCR was
carried out for 28 cycles of the following program: 1 min at 93°C, 1 min at 55°C, and 1 min at 72°C. For quantitation of relative
transcript levels, different amounts of RNA (100, 33, and 11 ng/µL)
for each induction time were subjected to the RT-PCR protocol described
above. Quantitation was as described by Giuliano et al. (1993).
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
|
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Effect of High Light Intensity on -Carotene Overproduction and
Lipid Deposition
-carotene formation when cultivated under high light intensities or
under sulfate or nitrate starvation (Ben Amotz et al., 1982). Figure
1 shows the response of the cells to a
high-light stress situation for 72 h. On the ultrastructural level
(Fig. 1D, 1 and 2), a reduction of thylakoid membranes, an increase in
starch granules, and a large accumulation of lipid droplets can be
observed. These droplets, when isolated by flotation, contained
triacylglycerol as the main lipid constituent (triacylglycerols, 63%;
polar lipids, 12%; carotene, 35%; Fried et al., 1982) and proved to
be red due to the presence of -carotene.
|
Inhibition of Fatty Acid Biosynthesis Reduces -carotene and acyl lipids
(as total fatty acids liberated upon saponification; Fig. 1, B and C);
both increase by a factor of 3 to 4 relative to protein. HPLC analysis
showed that -carotene was the major carotenoid, approximately 42%
being all-trans and 48% 9-cis -carotene; lutein was also present to a significant extent (data not shown). It
turned out that the only lipid class that increased during the 72 h of high-light-stress conditions was the triacylglycerols (Fig.
2A), which increased from 0.196 µmol
mg1 protein in noninduced cells at the beginning of
the experiment to 0.882 µmol mg1 protein in
induced cells after 72 h. This specific increase was further
confirmed by in vivo incubation of induced and noninduced cells with
NaH14CO3 for 48 h and
subsequent analysis of the distribution of radioactivity in the lipid
classes by two-dimensional TLC (Fig. 2B). The labeling of
triacylglycerol dramatically increased relative to the other lipids,
whereas the labeling of the plastid membrane galactolipids decreased
relatively, as expected.
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Figure 2.
Analysis of lipid classes formed after 48 h
of high-light treatment. A, TLC results. Std, Standard
(tripalmitoylglycerol); N.I, noninduced cells; I, induced cells. Car,
-carotene; TAG, triacylglycerol; PL + Chl, phospholipids + galactolipids + chlorophyll. B, Distribution of radioactivity from
NaH14CO3 (incubation time, 48 h) into
different lipid classes in control cells and in induced cells. Car,
-Carotene; GL, galactolipid; SL, sulfolipid; PL, phospholipid; and
Chl, chlorophyll.
-Carotene
Accumulation
-carotene as the final product
of the membrane-bound carotene biosynthetic pathway into a spatially
separated site may provide a mechanism to accelerate its biosynthesis.
To test for this possibility, cells were induced in the presence of two
differentially acting inhibitors of the lipid biosynthetic pathway:
sethoxydim, an inhibitor of the enzyme acetyl-CoA carboxylase (Burton
et al., 1987; Lichtenthaler, 1990), and cerulenin, which inhibits
-ketoacyl-(acyl carrier protein) synthases (Von Wettstein-Knowles,
1995).
).
Protein Abundance and Enzyme Activities
Transcript Levels for Phytoene Desaturase and an Early
Light-Induced-Like Protein during High-Light Treatment
In the present paper we addressed the question of whether, during
overproduction of carotenoids in the chloroplasts of the unicellular
alga D. bardawil, the formation of specialized lipophilic carotenoid sequestering structures represents a mere parallelism or
whether these structures exert a positive effect on carotenoid biosynthesis by pulling the pathway toward completion. On the basis of
the data presented here, we favor such a causal interdependence of
structure (lipid globule) formation and enhanced carotenoid biosynthesis. Sethoxydim and cerulenin, two inhibitors targeting different enzymes (ACCase and Received September 16, 1997;
accepted December 11, 1997.
Abbreviations:
IPP, isopentenyl diphosphate.
RT, reverse
transcriptase.
We are indebted to J. Ohlrogge for helpful discussions. We also
thank J. Hirschberg for providing D. bardawil phytoene
desaturase DNA-sequence information. Electron microscopy was performed
by V. Speth, whom we gratefully acknowledge. We would also like to thank R. Cassada, who corrected the English version of the manuscript.
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-carotene accumulation (Fig. 3); both rapidly
decreased with increasing inhibitor concentrations.
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Figure 3.
Cerulenin (A) and sethoxydim (B) inhibit
high-light-induced -carotene accumulation. Cells were induced by
high light and cultured in the absence (control cells) or presence of
different concentrations of inhibitors for 48 h and then analyzed.
, -Carotene; 
, total fatty acids. The data represent the means
of three values ± se. prot, Protein.
-carotene overproduction. In control
experiments performed with D. bardawil and N. pseudonarcissus chromoplasts no inhibitory effect on the carotene
biosynthetic pathway was noted for either compound in vitro (Table
I). The carotene pathway as a whole
seemed to be arrested, since in vivo there was no accumulation of
intermediates such as upstream carotenes or medium- and short-chain
isoprenoid diphosphates or alcohols, as analyzed using a combined
closed-loop stripping/GC analysis we described previously (Mettal et
al., 1988).
View this table:
Table I.
The inhibitors of lipid biosynthesis, sethoxydim and
cerulenin, do not interfere with carotene biosynthesis in vitro
Cell lysates (1.6 mg protein mL 1) from high-light-treated
D. bardawil cells were incubated for 4 h at 27°C with
18 kBq [1-14C]IPP in the presence of sethoxydim (50 µm) or cerulenin (8 µm). Radioactive
carotenes were isolated by TLC and individual carotenes were collected
from HPLC and quantified by liquid scintillation counting. S,
Sethoxydim; C, cerulenin.
-carotene biosynthesis are linked to each
other in a causal relationship. In molecular terms, this could mean
that a genetic induction of the carotene biosynthetic pathway is not
necessarily required to stimulate -carotene overproduction. Induction of the triacylglycerol biosynthetic pathway alone and the
formation of a sink consisting of sequestering lipid globules may be
sufficient to cause enhanced -carotene formation. To test this
hypothesis more closely, we monitored enzyme activities and investigated protein and mRNA abundance during the induction process.
et al., 1996) from N. pseudonarcissus
cross-reacted with the corresponding proteins in D. bardawil.
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Figure 4.
Western analysis showing the abundance of phytoene
synthase and phytoene desaturase during high-light treatment. Equal
amounts of protein (30 µg per lane) were separated and
electro-transferred as described in the experimental procedures.
Primary antibodies were anti-phytoene synthase antibodies (A) and
anti-phytoene desaturase antibodies (B). Secondary antibodies were
linked to horseradish peroxidase. Detection was performed using the
enhanced chemiluminescence system. C, The biotin-containing bands
showing an increase during high-light treatment represent an internal
control and were detected using alkaline phosphatase-conjugated
streptavidin. Ct, Control (N. pseudonarcissus
chromoplast extract).
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Figure 5.
Time course of the in vitro synthesis of acyl
lipids and prenyl lipids in D. bardawil cell lysates at
various times after onset of high-light induction of cells. A, The time
course of the incorporation of radioactivity into acyl lipids ( 
;
right scale) and prenyl lipids (; left scale) from
[1-14C]acetate and [1-14C]IPP,
respectively. At the times indicated, cells were lysed and incubated in
the presence of 37 kBq [1-14C]acetate or 28 kBq
[1-14C]IPP for 2 and 6 h, respectively. Data are the
means of three values. Vertical bars indicate ± se. B,
ACCase activity () and lipid accumulation (; measured as total
fatty acids) during high-light treatment of the cells. Values are
means ± se for three repetitions. prot, Protein.
, 1992; Page et al., 1994). A
remarkably strong increase in its specific activity was noted during
the first 12 h after induction, but it then declined rapidly.
Similar characteristics of ACCase activation have been reported
previously, e.g. during rapeseed formation (Turnham and Northcote,
1983). The ACCase activity measured in vitro was found to be 45%
inhibited by 50 mm sethoxydim (data not shown). Since, as
was shown above, triacylglycerols represent the main lipid constituent
of lipid globules, the specific activity of glycerol-3-phosphate
acyltransferases in vitro was measured using
[U-14C]glycerol-3-phosphate and oleoyl-CoA as
substrates. Indeed, 48 h after induction the specific activity of
this enzyme had concomitantly increased about 8-fold compared with
uninduced cells (not shown).
View larger version (44K):
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Figure 6.
RT-PCR analysis of phytoene desaturase
(pds) and CBR (cbr) mRNA expression in
D. bardawil during high-light induction. Total RNA was
isolated at different times after the onset of high-light treatment.
Left, RT-PCR reactions were performed with 100 (lanes 1), 33 (lanes 2),
and 11 ng (lanes 3) of total RNA. The 487-bp band is derived from
phytoene desaturase mRNA; 256-bp bands represent the CBR mRNA. Right,
The two transcripts were co-amplified using all four specific primers.
M, Marker ( 
-DNA digested using
EcoRI/HindIII).
-carotene accumulation.
). The phytoene
desaturase transcript levels (Fig. 6) remained constant during
high-light treatment, as was expected, whereas the CBR transcripts
increased steadily. This was also evident in an RT-PCR co-amplification
assay using all four primers; the bands, however, cannot be directly
compared in quantitative terms. In conclusion, although -carotene
synthesis is massively induced, both the mRNA and protein steady-state
concentrations for the biosynthetic enzymes remain constant.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
-ketoacyl-[acyl carrier protein] synthase, respectively) in the lipid biosynthetic pathway, both inhibited -carotene accumulation. The -carotene biosynthetic pathway itself proved to be insensitive toward these compounds. The
underlying chemical mechanisms of such an indirect acceleration of
-carotene synthesis are still unclear and may involve favorable chemical disequilibria created by such a lipophilic sink.
).
Thus, we propose that sequestering mechanisms contribute to the
regulation of carotenoid biosynthesis, at least in D. bardawil. And, since carotenoid overproduction is generally
accompanied by the development of specialized lipophilic structures, it
seems realistic to assume a more general role of such a
structure/activity relationship.
). It must be
noted that these isolated fibrils, like the D. bardawil
globules, did not show any carotenogenic activity in vitro; all
carotenoids bound here represented "dead-end products." It is
currently unknown what mechanism permits carotenoids to reach these
structures (globules, fibrils, or crystals), which are spatially
distinct from their site of formation in membranes. In this regard the
simplest case seems to be present in the so-called membranous type
chromoplast in N. pseudonarcissus petals, in which the
plastid membranes are proliferated so that the two sites (formation and
deposition) are identical.
et al., 1996; Schledz et al., 1996). The
enzyme ACCase, generally regarded as a key regulatory enzyme in acyl
lipid biosynthesis (for review, see Ohlrogge and Browse, 1995), was
induced in its activity. Additionally, the incorporation of acetate
into lipid products, the activities that drive globule formation,
typically preceded -carotene accumulation.
-carotene accumulation is at least to some degree a consequence of
triacylglycerol deposition. Similarly, during flower development (from
closed green bulb to open flower) in N. pseudonarcissus, no
strong increase of mRNA or of protein was noted for phytoene synthase
or phytoene desaturase, whereas carotenoids increased and membranes
proliferated, pointing to a similar regulatory mechanism (Al-Babili et
al., 1996; Schledz et al., 1996). However, there is a notable
up-regulation in N. pseudonarcissus flowers compared with
the first green leaves. This is paralleled by findings with C. annuum chromoplasts, in which phytoene synthase and phytoene desaturase mRNAs (Hugueney et al., 1992; Römer et al., 1993) increase between the mature green fruit and the first defined ripening
stage but then remain constant thereafter, not increasing subsequently
in parallel to the massive increase of carotenoids occurring during
later developmental stages (B. Camara, personal communication). In
contrast, the formation of fibrillin, the protein constituent of
fibrils, as well as the steady-state concentration of fibrillin mRNA,
increases strongly during the later stages of C. annuum
fruit ripening (Deruère et al., 1994).
Geranylgeranyl-diphosphate synthase behaves somewhat differently, being
induced in later stages (Kuntz et al., 1992). However, this
enzyme is not solely devoted to carotenoid biosynthesis and can
therefore not be readily considered so simply in this discussion.
) indicate that mRNA abundance
follows more closely color development, it must be taken into account
that a different strategy for carotenoid accumulation is used, namely
the formation of crystals. On thermodynamic grounds (no lipophilic
containment), this may be the most difficult mechanism; therefore, the
underlying genetic regulation may also be different.
-carotene accumulation.
); moreover, the
biochemical pathway for supplying the isopentenyl diphosphate
substrate, which may be crucial, is still obscure.
).
Dicot plants are known to possess the prokaryotic type in their
plastids (the site of fatty acid biosynthesis), whereas the eukaryotic type is present in this compartment in monocot plants (Sasaki et al.,
1995). The two ACCase types are also distinguished by their
differential susceptibility to the herbicide sethoxydim, which affects
selectively the eukaryotic form (for review, see Harwood, 1988). This
latter form of ACCase seems to be involved in the formation of
plastid lipid globules in D. bardawil during high-light treatment. To substantiate this result, we are currently investigating ACCase molecularly.
1
This study was supported in part by the
Deutscher Akademischer Austauschdienst (S.R.), by a European Molecular
Biology Organization long-term fellowship (P.H.), by the German-Israeli
Foundation (no. 214-240.12/91), and by the European Community BIOTECH
Program.
FOOTNOTES
*
Corresponding author; e-mail
kleinig{at}sun1.ruf.uni-freiburg.de; fax 49-761-203-2675.
ABBREVIATIONS
ACKNOWLEDGMENTS
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
-carotene accumulation in the halotolerant alga Dunaliella
bardawil. Plant Physiol 72: 593-597 -carotene in halotolerant algae: purification and characterization of -carotene rich globules from Dunaliella bardawil (Chlorophyceae).
J Phycol
18:
529-537
[Web of Science] -carotene
and phytoene in the alga Dunaliella bardawil. Plant Physiol
86: 1286-1291 
-carotene in
Capsicum chromoplasts. Eur J Biochem
209: 399-407 ,-carotene in thylakoids and in pigment globules in
Dunaliella. J Plant Physiol 143:
257-263 -carotene
biosynthesis in the alga Dunaliella bardawil. J Biol
Chem 266: 13698-13705 -ketoacyl-ACP synthases.
In
JC Kader,
P Mazliac,
eds, Plant Lipid Metabolism.
Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 61-71
Copyright Clearance Center: 0032-0889/98/116/1239/10
© 1998 American Society of Plant Physiologists
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-Carotene Synthesis Driven by Triacylglycerol
Deposition in the Unicellular Alga Dunaliella
bardawil
, Control cells;