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Plant Physiol. (1999) 119: 1279-1288
Cucumber Cotyledon Lipoxygenase during Postgerminative Growth.
Its Expression and Action on Lipid Bodies
Kenji Matsui*,
Kohko Hijiya,
Yutaka Tabuchi, and
Tadahiko Kajiwara
Department of Biological Chemistry, Faculty of Agriculture,
Yamaguchi University, Yamaguchi 753, Japan
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ABSTRACT |
In cucumber (Cucumis sativus), high lipoxygenase-1
(LOX-1) activity has been detected in the soluble fraction prepared
from cotyledons of germinating seeds, and the involvement of this
enzyme in lipid turnover has been suggested (K. Matsui, M. Irie, T. Kajiwara, A. Hatanaka [1992] Plant Sci 85: 23-32; I. Fuessner, C. Wasternack, H. Kindl, H. Kühn [1995] Proc Natl Acad Sci USA 92:
11849-11853). In this study we have investigated the expression of the
gene lox-1, corresponding to the LOX-1 enzyme. LOX-1
expression is highly coordinated with that of a typical glyoxysomal
enzyme, isocitrate lyase, during the postgerminative stage of cotyledon development. In contrast, although icl transcripts
accumulated in tissue during in vitro senescence, no accumulation of
lox-1 mRNA could be observed, suggesting that
lox-1 plays a specialized role in fat mobilization.
LOX-1 is also known to be a major lipid body protein. The partial
peptide sequences of purified LOX-1 and lipid body LOX-1 entirely
coincided with that deduced from the lox-1 cDNA
sequence. The data strongly suggest that LOX-1 and lipid body LOX-1 are
derived from a single gene and that LOX-1 can exist both in the cytosol
and on the lipid bodies. We constructed an in vitro oxygenation system
to address the mechanism of this dual localization and to investigate
the action of LOX-1 on lipids in the lipid bodies. LOX-1 cannot act on
the lipids in intact lipid bodies, although degradation of lipid body
proteins, either during seedling growth or by treatment with trypsin,
allows lipid bodies to become susceptible to LOX-1. We discuss the role
of LOX-1 in fat mobilization and its mechanism of action.
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INTRODUCTION |
LOXs (linoleate: oxygen oxidoreductase, EC
1.13.11.12) are widely distributed in plants and animals. They catalyze
the addition of molecular oxygen to fatty acids containing at least one
(Z),(Z)-1,4-pentadiene motif to give the
corresponding hydroperoxides. In animals LOXs use arachidonic acid as a
substrate and are involved in the formation of various regulatory
compounds such as leukotrienes and lipoxines (Samuelsson et al., 1987 ).
In plants LOXs oxygenate linoleic and linolenic acids at their 9 and 13 positions to give 9- and 13- hydroperoxy fatty acids, respectively. It
is known that many isozymes of LOX occur in various parts of plants,
and each is thought to play a different role in such processes as
development and response to wounding. It has been widely accepted that
plant LOXs have various physiological roles that are indispensable for
plant life (Siedow, 1991).
In oilseed plants such as cucumber (Cucumis sativus) and
sunflower, the storage lipids, which exist mostly as triacylglycerols, are tightly packed in subcellular organelles called lipid bodies (Huang, 1992). These spherical organelles are covered with a
phospholipid monolayer and extrinsic proteins. Lipid bodies are formed
when triacylglycerols are synthesized on the rough ER during seed
development. Oleosin, a "coat" protein surrounding the
triacylglycerol droplets, contributes to the stabilization of lipid
bodies during dehydration and seed dormancy (Napier et al., 1996).
However, once imbibition occurs and germination is triggered,
triacylglycerols are degraded to act as a carbon and energy source for
the developing seedling (Beevers, 1979). Acyl moieties of the stored
lipids are transported to glyoxysomes, where they are degraded by the
 -oxidation system to form acetyl-CoA. Resulting products are
eventually converted to oxaloacetate through isocitrate and malate
intermediates. This process operates via the glyoxylate cycle, a route
involving enzymes of the glyoxysomes.
The glyoxysomal enzymes ICL and MS have been well characterized
(Trelease et al., 1971; Weir et al., 1980). It is known that the genes
encoding these enzymes are highly expressed within several days after
germination in cucumber seedlings (Reynolds and Smith, 1995). It has
also been reported that LOX activity increases during the early stages
of seed germination in various plants (for review, see Roshal,
1996). Previously, we had found that LOX activity increased in the
soluble fraction prepared from cucumber cotyledons after germination
(Matsui et al., 1992). The developmental changes in LOX activity during
postgerminative growth of the cotyledons resemble changes in the level
of glyoxysomal enzymes. Accumulation of the enzyme in cucumber
cotyledons reaches levels as high as 2% to 5% of total protein. LOX
can oxygenate fatty acid moieties esterified in neutral lipids, as well
as free fatty acids (Matsui and Kajiwara, 1995). These observations
suggest that the enzyme is involved in lipid mobilization; if this is
the case, expression of the gene corresponding to LOX must be highly
coordinated with that of the glyoxysomal genes. In the present study we
investigated the expression of the lox-1 gene in cucumber
during the postgerminative growth of the cotyledons.
Feussner and Kindl (1992) reported that during the early stages of seed
germination LOX is the main lipid body protein in cucumber cotyledons.
Furthermore, by elucidating the structures of oxygenated lipids in
cucumber cotyledons, Feussner et al. (1997) proposed that the lipid
body LOX is involved in lipid mobilization. Considering these separate
data on LOXs found both in the soluble fraction and on lipid bodies, we
postulate that two different isozymes of LOX-1 may have almost the same
physiological role or that one isozyme may have distinct localizations.
To address this question, we isolated both of the LOXs and compared
their primary structures. We also constructed an in vitro oxidation system using purified LOX and lipid bodies isolated from cotyledons at
various stages of growth. Our investigation revealed how LOX on lipid
bodies acts on storage lipids.
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MATERIALS AND METHODS |
Plant Material
Cucumber (Cucumis sativus L. cv Suyo) seeds were soaked
for 12 h in tap water and sown on moistened paper towels. The
seeds were grown at 25°C in the dark or under a 16-h light/8-h dark photoperiod. For in vitro senescence, the cotyledons of 10-d-old seedlings were removed from their hypocotyls, placed abaxial side up on
a stack of moistened paper towels, and incubated in the dark at 25°C.
Fatty Acid Composition
We extracted total lipids from cucumber cotyledons using the
method of Bligh and Dyer (1959). The crude lipids were separated by TLC
(Kieselgel 60, Merck, Darmstadt, Germany) using
diisobutylketone:acetic acid:water (40:25:4, v/v) as the developing
solvent. Regions corresponding to neutral lipids were scraped off,
extracted with ether, mixed with heptadecanoic acid as an
internal standard, and then trans-esterified with 5% HCl in
methanol by heating at 100°C for 3 h. We used a gas
chromatograph (model GC-6A, Shimadzu, Columbia, MD) equipped with a 2%
Silar 5CP column (3 mm i.d. × 3 m height; Restek, Bellefonte, PA)
to quantitatively analyze the fatty acid methyl esters thus obtained.
The column temperature was 180°C to 210°C (2°C/min). The
respective ester was detected with a flame-ionization detector.
Purification of LOX
LOX was purified from cucumber cotyledons 5 DAG essentially as
described previously (Matsui et al., 1993) but with minor
modifications. We homogenized the cucumber cotyledons with cold acetone
and extracted LOX from the acetone powder and fractionated it with
ammonium sulfate. After dialysis, the enzyme solution was fractionated with a DEAE column (2.2 × 20.0 cm; Cellulofine A-500, Seikagaku, Tokyo) and then with a Butyl-Toyopearl 650M column (1.75 × 15.5 cm; Toyo Corp., Tokyo). The purified enzyme was stored at  20°C until use. In some experiments, we used LOX expressed in
Escherichia coli cells. E. coli Y1090 ( pMc9)
cells transformed with pTrc99A (Pharmacia) and containing a cucumber
cotyledon LOX cDNA (lox-1) were grown at 30°C for 4 h
in 2 L of Luria-Bertani broth, supplemented with 50 µg
mL 1 ampicillin.
Isopropyl-1-thio--D-galactoside was then added
at a final concentration of 0.5 mM, and the
culture was incubated at 30°C for an additional 6 h. The cells
were harvested by centrifugation at 6,000 rpm for 10 min, resuspended
in 80 mL of lysis buffer (50 mM Tris-Cl, pH 7.5, 1 mM EDTA, and 0.1 mM
NaCl), and repelleted by centrifugation at 6,000 rpm. The washed cells
were resuspended in 120 mL of lysis buffer containing 0.13 mM PMSF. Lysozyme was added at a concentration of
0.1 mg mL1, and the solution was incubated on ice for 20 min. Finally, the cells were disrupted by a French press (AMINCO,
Spectronic Instruments, Rochester, NY). The cell homogenate was
centrifuged at 10,000 rpm for 10 min to give a crude extract. The
extract was fractionated with ammonium sulfate, dialyzed, and column
purified with a DEAE column.
We determined LOX activity polarographically with a Clark-type oxygen
electrode (Yellow Springs Instruments, Yellow Springs, OH) at 25°C
(Matsui and Kajiwara, 1995). With free fatty acid as the substrate, 0.1 M sodium phosphate, pH 6.3, was the buffer; whereas with
triglycerides or a lipid body suspension as the substrate, 0.1 M Tris-Cl, pH 8.5, was the buffer. The activity (1 kat) is defined as the quantity of the enzyme catalyzing the consumption of 1 mol O2 s1 at 25°C.
In Situ Digestion of LOX
We separated the purified cucumber LOX with SDS-PAGE and then
transferred it electrophoretically to a PVDF membrane (Millipore). The
membrane was briefly stained using Ponceau S, and the portion containing LOX was cut out. Fifty milliliters of 0.5% PVP 40 (Sigma) in 0.1 M acetic acid was added to the membrane strip and
shaken at 37°C for 30 min. After the membrane was washed, it was
equilibrated with 50 mL of 0.1 M Tris-Cl, pH 9.0. The
protein on the membrane was digested with lysil endopeptidase (Wako
Pure Chemicals, Tokyo) and then separated with reversed-phase HPLC. We
separated the peptide fragments with an HPLC system equipped with a
C18 column (4.6 mm i.d. × 250 mm; CosmoSIL,
Nakalai Tesque, Kyoto, Japan). Peptide fragments were eluted with a
linear gradient from 10% acetonitrile and 0.1% trifluoroacetic acid
in water to 50% acetonitrile and 0.1% trifluoroacetic acid in water
for 70 min. The flow rate was 0.5 mL min1. We
used a UV detector (model L-4000, Hitachi, Tokyo) for detection at 215 nm. The separated peptides were dried under nitrogen at 60°C,
dissolved in 10 µL of formic acid and 20 µL of distilled water, and
then applied to a protein sequencer (model PSQ-1, Hitachi, Tokyo).
Preparation of Lipid Bodies
Cucumber cotyledons (100 pairs) excised from seedlings grown for 1 to 6 d were homogenized with 10 mL of homogenization buffer (0.15 M Tris-Cl, pH 7.5, 15% Suc, 10 mM KCl, 1.5 mM EDTA, 0.1 mM MgSO4, 5 mM dithioerythritol, and 10 mM sodium
ascorbate) in a mortar with a pestle using sea sand. The homogenate was
centrifuged at 5,000 rpm for 5 min at 4°C. The resulting supernatant
was centrifuged at 40,000 rpm for 1 h at 4°C in a swinging
bucket rotor. The lipid layer that formed on the upper surface was
carefully collected with a spatula and resuspended in 2 mL of 0.1 M NaHCO3. This suspension was
centrifuged again at 40,000 rpm for 1 h at 4°C, and the lipid layer was collected and resuspended in 2 mL of the homogenization buffer. The suspension of lipid bodies was stored at 20°C until further use.
The lipid body suspension was incubated for 2 h at 25°C in 60 mL
of a 0.1 M Tris-Cl buffer, pH 8.0, containing various
concentrations of trypsin (Sigma). We then added PMSF at a final
concentration of 0.1 mM to inactivate the trypsin. Part of
the reaction mixture was heated immediately at 100°C for 3 min in an
SDS sample buffer for SDS-PAGE analysis. We used the remaining solution
immediately as a substrate for LOX.
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RESULTS |
Developmental Changes in Cucumber Cotyledon Fats
As shown in Table I, linoleic acids
are the main fatty acid component in stored fats, making up as much as
65% of total fats in the cotyledons of dry cucumber seeds. The amount
of total fats started to decrease from 2 DAG. The decrease was rapid,
and by 6 DAG only 3% of the initial fats could be detected. This
indicates that under these growth conditions turnover of the stored
fats occurred rapidly from 3 to 6 DAG. The rapid degradation of the stored fats started from the time that radicles appeared and lasted until the cotyledons fully expanded (Fig.
1A). During this period, protein
composition in the cotyledons also changed rapidly (Fig. 1B).
Degradation of seed storage proteins preserved in dry seeds started to
appear from 3 DAG (Yamaguchi et al., 1996).
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Table I.
Fatty acid composition in the neutral lipid fraction
of cucumber cotyledons
Values in parentheses are the percentages of total neutral lipids.
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| Figure 1.
Developmental changes in morphology (A), protein
profiles of total soluble protein (B), and lipid body fraction (C),
during early germinating stages of cucumber grown under a 14-h
light/10-h dark photoperiod. For total soluble protein, proteins
equivalent to 0.025 cotyledon pairs were loaded in each lane, and for
lipid body fractions 0.25 cotyledon pairs were used.
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When the seed storage proteins had almost disappeared, the accumulation
of the large subunit of Rubisco became evident, clearly representing a
transition of the metabolism of cucumber cotyledons from heterotrophic
growth, which is dependent on energy sources preserved in dry seeds, to
photoautotrophic growth, which is dependent on photosynthetic carbon
assimilation. During this transition, a protein of about 96 kD
appeared. Immunological staining of the protein indicated that it is
LOX (Matsui et al., 1992). At d 4 LOX accounted for as much as 2% to
5% of total soluble proteins. Most LOX activity could be found in the
soluble fraction of the cotyledon homogenate. This developmental time
course of LOX accumulation in the soluble fraction is almost identical
to that of the enzymatic activity (Matsui et al., 1992).
LOX activity can also be detected in the microsomal membrane fraction,
as reported by Fuessner and Kindl (1994); however, this is equivalent
to less than 1% of the activity in the soluble fraction. From the
specific activity of the LOX purified from the soluble fraction of
cucumber cotyledons at d 5 (Matsui et al., 1993), the amount of LOX in
the cotyledons was calculated to be about 5% of the total protein.
This is almost equivalent to the amount estimated by SDS-PAGE analysis.
High expression of LOX in cucumber cotyledons was observed when the
drastic metabolic change was occurring, i.e. when the stored fats
rapidly degraded. Tight correlation between the developmental changes
of LOX and the turnover of stored fats suggests that LOX takes part in
fat degradation.
Detection of LOX Genes in Cucumber
It is well known that during fat mobilization the activities of
glyoxysomal enzymes such as ICL and MS are enhanced to drive the
glyoxylate cycle (Beevers, 1979). If cucumber cotyledon LOXs are also
involved in fat mobilization, then their expression should correlate
with that of the glyoxysomal enzymes. Most plants have several isozymes
of LOX (Siedow, 1991), and it is difficult to discriminate each isozyme
using only an activity assay. To overcome this limitation, it was
necessary to investigate LOX expression with a gene-specific detection
system. Figure 2A shows the results of
Southern analysis, in which a probe covering the coding region of
cucumber cotyledon LOX cDNA, lox-1 (accession no. U25058, Matsui et al., 1995) produced a complicated picture of the LOX gene
family. Matsui et al. (1998) reported the occurrence of a LOX gene
other than lox-1, whose expression is specific to cucumber roots. However, the complicated Southern-blot pattern cannot be explained even if these two genes are taken into account. There must be
more than two LOX genes in cucumber. In contrast, a lox-1 probe derived from the 3 -noncoding region detected only one band on
the blot, which indicates that the probe detected specifically the
lox-1 sequence (Fig. 2B).
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| Figure 2.
Genomic Southern analysis. About 10 µg of
genomic DNA from cucumber was digested with HincII (lane
1), XbaI (lane 2), NdeI (lane 3),
NcoI (lane 4), HindIII (lane 5), and
EcoRI (lane 6) and then separated on a 0.6% agarose
gel. As a probe, an EcoRI fragment of
lox-1 (A) or an EcoRI 3-end fragment (B)
was used. A schematic diagram of lox-1 and templates
used for the preparation of probes are shown in C.
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With the 3 probe, northern analysis revealed that lox-1 is
specifically expressed in cotyledons and hypocotyls in the seedlings (Fig. 3A). Matsui et al. (1988) reported
that the properties of LOX activity in the hypocotyls are almost the
same as those in the cotyledons. With the internal probe, expression of
LOX in the roots and in reproductive organs such as buds, flowers, and immature fruits could be detected in addition to the cotyledons and
hypocotyls (Fig. 3B). Expression of LOX genes in pea carpels (Rodriguez-Concepcion and Beltran, 1995) and in Arabidopsis
inflorescences (Bell and Mullet, 1993) has been reported, and their
involvement in a reproductive process has been proposed. Expression of
LOX in fruits has been reported with various plants, including tomatoes (Ferrie et al., 1994) and bell peppers (Matsui et al., 1997). LOX
activity has also been detected in cucumber fruits, and it appeared to
be involved in the formation of volatile compounds such as
(2E)-nonenal or n-hexanal; the former is known to
be an essential determinant of cucumber flavor (Galliard et al., 1976).
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| Figure 3.
Expression of LOX mRNA in cucumber tissues. Blots
were hybridized with a lox-1 3 probe (A) or a
lox-1 internal probe (B). Nucleic acids were loaded at
10 µg/lane. Lanes 1, Cotyledons of 4-d-old seedlings; lanes 2, hypocotyls of 4-d-old seedlings; lanes 3, roots of 4-d-old seedlings;
lanes 4, mature leaves; lanes 5, stems; lanes 6, tendrils; lanes 7, mature roots; lanes 8, buds; lanes 9, male flowers; lanes 10, female
flowers; lanes 11, immature fruits less than 3 cm in length; and lanes
12, immature fruits 4 to 8 cm in length.
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Expression of the lox-1 Gene
We investigated the expression of the lox-1 gene in
cotyledons during seed germination in the light and in the dark. For
comparison, the pattern of expression of the icl gene
(Reynolds and Smith, 1995) was also investigated. The results (Fig.
4, A and C) show that the levels of
lox-1 and icl mRNA increased and decreased in
parallel. In the light, accumulation of lox-1 and
icl mRNAs was already evident by d 3. Both rapidly declined
to undetectable amounts by d 5. This rapid decline occurred
concomitantly with the greening of the cotyledons, as photosynthesis
became established. Figure 1A shows the accumulation of the large
subunit of Rubisco. In the dark, rapid increases in the amounts of both
mRNAs lasted until d 4 and remained at a high level until d 7. Both
declined slowly and were still detectable at d 8. When the dark-grown
seedlings were transferred to the light, the accumulated mRNAs rapidly
disappeared. It is notable that expression of the lox-1 and
icl genes were strictly coordinated. This may mean that they
share at least one similar cis-acting DNA sequence that
regulates their transcription. When we used the internal probe for the
detection of LOX mRNAs in the cucumber cotyledons, we observed almost
the same pattern as with the 3 probe (Fig. 4B). However, careful
examination of the blot shows that low but significant expression of
the LOX gene(s) occurred in green cotyledons even after d 5, when
lox-1 mRNA was undetectable (Fig. 4B). This was also
observed after the transfer of the dark-grown seedlings to the light.
Degradation of lox-1 mRNA was evident in northern analyses,
especially when the internal probe was used. Because distinct bands can
be seen with the icl probe, such degradation might account
for the instability of lox mRNA. Further experiments are
needed to confirm this.
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| Figure 4.
Accumulation of LOX and icl
transcripts during early germinating stages of cucumber cotyledons in
the light or dark. Cucumber seeds were germinated on vermiculite and
then grown under a 14-h light/10-h dark photoperiod or in complete
darkness. A portion of cucumber seedlings grown for 5 d in the
dark was transferred to the 14-h light/10-h dark photoperiod. Twenty
micrograms of total RNA isolated from each group of cotyledons was
separated with a denaturing gel, transferred to a nylon membrane, and
sequentially probed with the lox-1 3 probe (A), the
lox-1 internal probe (B), and the icl
probe (C). A and B, Some degradation of lox-1 mRNA is
evident. Because distinct bands can be seen with the icl
probe (as in C), such degradation does not reflect a problem in RNA
isolation. D to L, Dark to light transition period.
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Expression of the LOX Gene during in Vitro Senescence
McLaughlin and Smith (1994) reported that the glyoxylate-cycle
enzymes ICL and MS accumulate when cotyledons are detached from
cucumber plants and are kept in the dark. The induction of these
glyoxysomal enzymes occurs before degradation of chloroplast lipids,
suggesting that the induction is not related to the turnover of lipids.
Although the functions of these two enzymes in the detached cotyledons
have not yet been elucidated, their expression is thought to be
regulated by the depletion of a product of Suc metabolism resulting
from the absence of photosynthesis (McLaughlin and Smith, 1994). To
reveal whether lox-1 expression correlates with that of
icl during in vitro senescence, we analyzed the amounts of
lox-1 and icl transcripts in cotyledons detached
from d-10 cucumber plants kept in the dark (Fig.
5). As reported, icl
transcripts started to accumulate from d 1 after excision and continued
to increase up to 10 d after the treatment. No induction of
lox-1 expression was observed with the lox-1 3
probe. On the other hand, the internal probe revealed the accumulation
of LOX transcripts in the cotyledons 1 d after excision; although
thereafter they disappeared. Bell and Mullet (1993) reported the
induction of LOX gene expression in Arabidopsis upon wounding. The
accumulation of LOX transcripts detected with the internal probe may
represent such a wound-inducible LOX gene.
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| Figure 5.
Accumulation of LOX and icl
transcripts during in vitro senescence of cucumber cotyledons.
Cotyledons were excised from cucumber seedlings grown for 10 d
under a 14-h light/10-h dark photoperiod and then kept in the dark for
the number of days indicated. Total RNA (20 µg) isolated from the
cotyledons was loaded in each lane, separated with a denaturing gel,
transferred to a nylon membrane, and then sequentially probed with the
lox-1 3 probe (A), the lox-1 internal
probe (B), and the icl probe (C).
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Sequence Comparison of LOXs Purified from Soluble and Lipid Body
Fractions
The observations presented above suggest that the lox-1
gene product takes part in fat mobilization during the early stages of
cucumber seed germination. Enzymatic analysis of LOX-1 indicated that
the enzyme can act on acyl groups esterified in neutral lipids (Matsui
and Kajiwara, 1995). This property led us to suggest that LOX-1 exerts
its function on lipid bodies. On the other hand, Feussner and Kindl
(1992) reported that LOX is the main lipid body protein in cucumber
during the period of triacylglyceride mobilization. Recently, these
same investigators reported the primary sequence of the lipid body LOX
gene cslblox (accession no. X92890, Hohne et al., 1996). The
nucleotide sequences of lox-1 and cslblox share
almost complete identity, even within the noncoding regions. When the
two sequences (note that U25058 was recently updated) are compared,
only three differences can be found at the nucleotide level (excluding
the extreme 3 end). To determine whether the two types of LOX are
isozymes encoded by different genes or are the same molecule encoded by
a single gene, we isolated both the soluble and the
lipid-body-associated LOXs and determined their internal amino acid
sequences. As shown in Figure 6, all of
the sequences determined with the soluble LOX or the lipid body LOX are
completely identical to the amino acid sequence deduced from
lox-1. This indicates that the soluble LOX and the lipid
body LOX may be derived from the same gene, even though their
intracellular localization differs.
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| Figure 6.
Identity between amino acid sequences determined
with soluble LOX and lipid body LOX purified from cucumber cotyledons.
The amino acid sequence deduced from the lox-1 cDNA
sequence (accession no. U25058) is shown. The sequences determined with
peptides derived from soluble LOX are underlined; those from lipid body
LOX are double underlined. Three differences can be found between
lox-1 and cslblox (accession no. X92890)
at the nucleotide level. Two of the three differences produce one amino
acid substitution (Ser to Ala), as shown at position 491, although the
amino acid sequence around Ser-491, namely IELUSLPHP, is
highly conserved within plant LOXs and no such substitution can be
found. The other difference is silent, and we do not know whether the
difference is caused by a sequencing artifact or by the different
varieties used as the gene source.
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In Vitro Oxygenation of Lipid Bodies by LOX-1
LOX-1 can oxidize fatty acids esterified in neutral lipids (Matsui
and Kajiwara, 1995). Recently, Fuessner et al. (1997) reported almost the same observation with LOX isolated from lipid bodies. However, it remains unclear whether LOX-1 actually acts on lipids in
lipid bodies. An in vitro oxygenation system was constructed to
investigate whether fats in lipid bodies can actually be substrates for
LOX-1. When lipid bodies alone were incubated under the assay conditions used here, little oxygen consumption was observed, although
binding of LOX to lipid bodies was evident. When lipid bodies prepared
from 1-d-old cotyledons were used as substrates for LOX-1, no activity
was found (Fig. 7). However, LOX-1 did oxidize the fat constituents when lipid bodies from the d-2 or later
cotyledons were used. As the cotyledons grew, LOX-1 activity on lipid
bodies increased. As shown in Figure 1C, proteins associated with lipid
bodies, such as oleosins, started to degrade at d 2 and almost
completely disappeared by d 5. Fatty acid composition of lipids in
lipid bodies was almost always independent of the stage of the
cotyledons (data not shown). These results indicate that LOX-1 can act
on fats sorted into lipid bodies only after lipid body proteins such as
oleosins are degraded. The same tendency was also observed with LOX-1
expressed in E. coli. Association of LOX-1 with lipid bodies
starts to take place at the same time as the degradation of the other
lipid body proteins.
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| Figure 7.
Oxygenation by LOX-1 of lipids in lipid bodies
isolated at different times after germination. Activity of LOX-1 on
lipid bodies was determined polarographically. Lipid bodies
corresponding to 8.3 µM equivalents of linoleic acid were
added to the reaction mixture (1.75 mL total). For comparison,
activities with linoleic acid and trilinolein are also shown.
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Trypsin Digestion of the Proteins Coating Lipid Bodies
Lipid body proteins such as oleosins are known to stabilize the
morphology of lipid bodies and to keep them intact during the
desiccation of seeds (Huang, 1992). The results shown above suggest
that lipid body proteins disturb the association of LOX-1 with these
organelles and thus prevent their oxygenation. To confirm this
hypothesis, lipid body proteins were digested using trypsin. As shown
in Figure 8A, LOX-1 was only slightly
active on the lipid bodies prepared from d-2 cotyledons that had their
coating proteins mostly intact. After partial trypsin digestion,
however, LOX-1 was able to oxidize fats in lipid bodies. The higher the
trypsin concentration, the higher the LOX activity. With a trypsin
concentration greater than 0.3 mg mL1, activity
was much higher than that observed when trilinolein emulsion was used
as a substrate. Protein profiles of trypsin-treated lipid bodies are
shown in Figure 8B. Treatment with low amounts of trypsin resulted in
the formation of distinct bands of degraded lipid body proteins.
It has been reported that oleosins are composed of three domains
(Huang, 1992). The fragments remaining even after treatment with
trypsin might represent the subunit protected by fats. This indicates
that removing the protein moieties covering the surface of oil bodies
may allow LOX-1 access to the lipids.
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| Figure 8.
Activity of LOX-1 on the lipids in lipid bodies
treated with trypsin. A, Lipid bodies isolated from 2-d-old cotyledons
were treated with a given amount of trypsin and then provided as a
substrate for LOX-1. B, Degradation of the lipid body proteins.
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DISCUSSION |
Plants, especially oilseed plants such as cucumber, use seed
storage lipids to provide energy for their postgerminative growth. The
process by which this occurs had been thought to be the following: (a)
acyl moieties of neutral lipids stored in lipid bodies are transported
to glyoxysomes and converted to acyl-CoAs there; (b) the acyl-CoAs are
then oxidized by the -oxidation system in glyoxysomes; (c) the
acetyl-CoAs formed through -oxidation go into the glyoxylate cycle
in glyoxysomes; and, finally, (d) the carbon source is transconverted into Glc via gluconeogenesis (Beevers, 1979). However, the first committed step of the turnover of the lipids has not yet been well
established. It has been assumed that lipid mobilization is initiated
by the liberation of free fatty acids from storage lipids, but a lipase
activity thought to be responsible for fatty acid liberation from lipid
bodies has not yet been detected in cucumber (Fuessner et al., 1997).
How the fats stored in lipid bodies are hydrolyzed to form acyl-CoA and
how they are delivered to glyoxysomes remains an enigma.
We have detected high LOX activity in germinating cucumber cotyledons
and have isolated a cDNA clone (lox-1) corresponding to the
activity (Matsui et al., 1992, 1995). Independently, Fuessner and Kindl
(1992) found that LOX is the main lipid body protein in cucumber
cotyledons. The primary sequence of the corresponding cDNA clone,
cslblox, has been published (Hohne et al., 1996). In the
present study we present evidence that these two LOXs are synthesized
from the same gene, lox-1. We have shown that (a) the
primary sequences of lox-1 and cslblox are almost
entirely identical, even within the 3- and 5-noncoding regions; and
(b) partial amino acid sequences of the LOXs purified from both the soluble and lipid body fractions of cucumber cotyledons are completely identical to the sequence deduced from lox-1. This indicates
that LOX-1 can exist in the cytosol as a soluble protein and
simultaneously on the lipid body surface as a membrane-associated
protein.
Previously, we showed that LOX-1 binds to the lipid/water interface
irreversibly (Matsui and Kajiwara, 1995). LOX-1 has an approximately
40-amino acid extension in its primary sequence that cannot be found in
other plant LOXs. This N-terminal extension shows no homology to any
known signal sequences. A prediction of its secondary structure
indicates that it forms an amphipathic  -helix (data not shown). Such
a conformation is also known for the C-terminal region of oleosins, and
its amphipathicity is believed to allow the -helix to interact with
the surface of oil bodies (Huang, 1992). The remainder of LOX-1 is
highly homologous to the other plant LOXs, most of which are soluble
proteins. In this context, it can be assumed that LOX-1 usually exists
as a soluble protein, but once it finds a lipid/water interface with
specific physical properties, LOX-1 associates with it through its
N-terminal extension. Such an interface must be specifically formed by
the lipid body surface after degradation of the lipid body proteins.
This study demonstrates that LOX-1 can act on the neutral lipids in
lipid bodies only after lipid body proteins such as oleosins are
degraded. LOX-1 can oxygenate lipids upon its irreversible binding to
the lipid/water interface (Matsui and Kajiwara, 1995), which means that
it can associate with the lipid body surface. The surface of a lipid
body is thought to be entirely covered with oleosins (Huang, 1992), and
therefore LOX-1 cannot find sites to associate with on intact lipid
bodies of cucumber cotyledons when fat mobilization has not yet
started. As the degradation of oleosins proceeds, the naked surface of
lipid bodies, composed of a half-membrane of phospholipids (and
probably partly of neutral lipids) would be exposed to the cytosol. As
a result of this exposure, LOX-1 can bind to the lipid bodies. We
provide strong support for this theory, showing that in vitro
degradation of the lipid body proteins with trypsin made lipid bodies
accessible to LOX-1.
Oleosins stabilize lipid bodies not only by covering their surface but
also by providing steric hindrance and electronegative repulsion. In a
germinating sesame seed, Tzen et al. (1997) observed oil bodies that
are larger than those found in a dry seed. Coalescence of lipid bodies
is also observed after trypsin digestion of oleosins. It is conceivable
that the physical state of the surface of coalesced lipid bodies is
highly modified. LOX-1 might recognize such a specific interface and
associate with it. Unexpectedly, the incubation of lipid bodies alone
exerted little oxygen consumption, even if LOXs attached to their
surfaces. Inactivation of LOX might occur during the preparation of
lipid bodies. Many examples of the inactivation of LOX after incubation
with its substrate have been reported (Matsui et al., 1998, and refs.
therein).
Feussner et al. (1995, 1997) analyzed products formed from the neutral
lipids in lipid bodies and suggested a role for LOX in lipid
mobilization during seed germination of cucumber and other plants.
Lipid mobilization is a highly organized system involving
-oxidation, the glyoxylate cycle, and gluconeogenesis and takes
place at a distinct time during the postgerminative growth of
seedlings. Therefore, the expression of lox-1 must be coordinated with other genes involved in lipid mobilization if LOX-1 is
actually involved in this process. Regulation of the enzymes in the
glyoxylate cycle, e.g. ICL and MS, has been intensely investigated
(Trelease et al., 1971; Weir et al., 1980). When the dry seeds
germinate, their enzyme activities increase from very low amounts to
peak levels after a few days of growth. After reaching their highest
level of activity, they decrease rapidly, concomitant with seedling
exposure to light and the establishment of photosynthesis. As shown in
this study, expression of lox-1 is highly coordinated with
icl in cucumber cotyledons in the postgerminative stage.
This suggests that lox-1 and icl share a
cis-acting element for the germination response (De Bellis
et al., 1997). Kim and Smith (1994) previously argued that the
germination response was probably not mediated by sugars, but the rapid
disappearance of the lox-1 and icl mRNAs in the
greening cotyledons is now thought to be caused by the enhanced flux of
some kind of sugar metabolite (Graham et al., 1994). In this context,
both genes again might share a cis-acting element that
suppresses their expression in response to a carbon
catabolite. Jang and Sheen (1997) have reported that the
sugar-repression-signaling pathways require hexokinase-mediated sugar
sensing in yeast and plants. On the other hand, such a coordination between lox-1 and icl was not observed during in
vitro senescence of the cotyledons. During in vitro senescence the flux
of sugar metabolites is thought to be lowered through the lack of
photosynthesis, which causes expression of icl in
cucumber (McLaughlin and Smith, 1994). Most likely, lox-1
does not have the cis-acting element responsive to the
sugar-activation pathway.
Kim and Smith (1994) have reported an expression profile similar to
that of lox-1 for the microbody NAD-malate dehydrogenase gene in cucumber. Most investigators believe this enzyme is involved in
the glyoxylate cycle. Like icl, the corresponding gene,
mdh, showed a typical germination response; however,
expression of the mdh gene was not activated by incubating
detached green cotyledons in the dark; nor was it affected by exogenous
Suc in the incubation medium. It should be noted that during the early
stages of in vitro senescence lipid turnover was not initiated, and an
alternative role for ICL during this period is expected (McLaughlin and
Smith, 1994). The synthesis of ICL is neither tissue specific nor
confined to the fat-storing tissues of germinating oilseeds; rather,
the physiological or metabolic state of the tissue may control the expression of this gene. Lox-1 gene expression, in contrast,
is confined to the fat-storing tissues of germinating oilseeds. To summarize, the expression of lox-1 is under strict
developmental regulation that is coordinated with that of glyoxysomal
genes, but this coordination is evident only in cotyledons undergoing postgerminative growth. This encourages us to speculate that LOX-1 plays an important role in lipid mobilization.
In conclusion, the following scheme can be proposed for the first
committed step of fat mobilization: in cucumber cotyledons undergoing
postgerminative growth, processing of lipid body proteins proceeds
first. At the same time, lox-1 is expressed in coordination with the expression of the glyoxysomal enzymes. LOX-1 thus formed starts to associate with lipid bodies. The absorbed LOX-1 oxygenates phospholipids and neutral lipids in lipid bodies to form oxidized lipids (Feussner et al., 1997). The oxidized lipids may be hydrolyzed to form oxidized fatty acids by a still-unknown lipase. About two-thirds of the acyl moieties in neutral lipids consist of linoleic acids; therefore, this action would leave essentially monoacylglycerols and diacylglycerols containing saturated and monounsaturated fatty acids. These are not substrates for LOX-1; however, accumulation of
these partially hydrolyzed lipids would lower the integrity of lipid
bodies further, which might make these lipids much more susceptible to
catabolism.
Currently, the most important missing part of this hypothesis is the
supporting evidence for a hydroperoxide-specific lipase. Such lipase
activity has been detected in rat liver (Kambayashi et al., 1997) and
in several plants (Stahl et al., 1995). Another question also arises
concerning LOX-1 levels and localization. A rough calculation indicates
that even in cotyledons that are intensively degrading the stored
lipids about 90% of LOX-1 remains in the soluble fraction and only
10% is associated with lipid bodies. This is probably due to the
limited number of attachment sites that are accessible to LOX-1. LOX-1
in the soluble fraction does not seem to oxygenate the storage lipids
in lipid bodies; therefore, it may have another role in the cytosol,
such as transporting lipid constituents to glyoxysomes.
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FOOTNOTES |
*
Corresponding author; e-mail
matsui{at}agr.yamaguchi-u.ac.jp; fax 81-839-33-5820.
Received October 1, 1998;
accepted December 9, 1998.
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ABBREVIATIONS |
Abbreviations:
DAG, days after germination.
ICL, isocitrate
lyase.
LOX, lipoxygenase.
MS, malate synthase.
X:Y, a fatty acyl group
containing X carbon atoms and Y cis double bonds.
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ACKNOWLEDGMENTS |
The authors wish to thank Dr. Jack Wilkinson for his critical
reading of the manuscript and Dr. Steven M. Smith for providing icl
cDNA of cucumber.
| |
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Abstract
Introduction