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Plant Physiol. (1998) 117: 923-930
A New Class of Arabidopsis Mutants with Reduced Hexadecatrienoic
Acid Fatty Acid Levels1
Martine Miquel*,
Claude Cassagne, and
John Browse
Laboratoire Biogenèse Membranaire, Université Victor
Segalen (Centre National de la Recherche Scientifique, Unité
Mixte de Recherche 5544), 146 rue Léo Saignat, Case 92, 33076 Bordeaux cedex, France (M.M., C.C.); and Institute of Biological
Chemistry, Washington State University, Pullman, Washington 99164-6340
(J.B.)
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ABSTRACT |
Chloroplast glycerolipids in a number
of higher-plant species, including Arabidopsis thaliana,
are synthesized by two distinct pathways termed the prokaryotic and
eukaryotic pathways. The molecules of galactolipids produced by the
prokaryotic pathway contain substantial amounts of hexadecatrienoic
acid fatty acid. Here we describe a new class of mutants, designated
gly1, with reduced levels of hexadecatrienoic acid.
Lipid fatty acid profiles indicated that gly1 mutants
exhibited a reduced carbon flux through the prokaryotic pathway that
was compensated for by an increased carbon flux through the eukaryotic
pathway. Genetic and biochemical approaches revealed that the
gly1 phenotype could not be explained by a deficiency in
the enzymes of the prokaryotic pathway. The flux of fatty acids into
the prokaryotic pathway is sensitive to changes in glycerol-3-phosphate (G3P) availability, and the chloroplast G3P pool can be increased by
exogenous application of glycerol to leaves. Exogenous glycerol treatment of gly1 plants allowed chemical
complementation of the mutant phenotype. These results are consistent
with a mutant lesion affecting the G3P supply within the chloroplast.
The gly1 mutants may therefore help in determining the
pathway for synthesis of chloroplast G3P.
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INTRODUCTION |
Higher plants possess two distinct pathways for the synthesis of
chloroplast glycerolipids in leaf cells (Roughan et al., 1980 ; Browse
and Somerville, 1991). The chloroplasts or the plastids of the cell are
the sole site of de novo fatty acid synthesis (Ohlrogge et al., 1991).
The final products of fatty acid synthesis and of the soluble stearoyl
ACP desaturase are 16:0-ACP and 18:1-ACP (McKeon and Stumpf, 1982;
Shanklin and Somerville, 1991). These either enter the prokaryotic
pathway of the chloroplast inner envelope to produce chloroplastic
lipids or they are hydrolyzed to free fatty acids that are exported
through the plastid envelope to the cytoplasm as CoA thioesters, thus
initiating the eukaryotic pathway. Because of the specificities of the
plastid acyltransferases for certain acyl-ACP substrates (Frentzen,
1993), the PA made by the prokaryotic pathway has 16:0 at the
sn-2 position and, in most cases, 18:1 at the
sn-1 position. This PA is used for the synthesis of PG or is
converted to DAG by a PAPase (Joyard and Douce, 1977). This DAG
pool is the precursor for the synthesis of MGD, DGD, and SL, the major
plastid membrane lipids (Joyard et al., 1993). The PA synthesized in
the ER by a different set of acyltransferases than the plastid isozymes
is characteristically enriched in 18-carbon fatty acids at the
sn-2 position; 16:0, when present, is confined to the
sn-1 position. This PA is used to produce phospholipids such
as PC, PE, and PI, which are characteristic of the various
extrachloroplastic membranes of the cell. In addition, a portion of PC
produced by the eukaryotic pathway is returned to the chloroplast and
used in the production of chloroplast lipids (Browse and Somerville,
1991).
In the majority of higher plants, PG is the only product of the
prokaryotic pathway, and the remaining chloroplast lipids are
synthesized entirely by the eukaryotic pathway (Browse et al., 1986b).
However, in a number of species, including Arabidopsis, both
pathways contribute about equally to the synthesis of MGD, DGD, and SL
(Browse and Somerville, 1991), and the leaf lipids of such plants
characteristically contain substantial amounts of 16:3, which is found
only at the sn-2 position of galactolipid molecules produced
by the prokaryotic pathway. These species have been termed 16:3 plants
to distinguish them from 18:3 plants, the galactolipids of which
contain predominantly  -linolenate (Jamieson and Reed, 1971; Browse
and Somerville, 1991).
Mutants of Arabidopsis with altered fatty acid composition have been
isolated (Browse and Somerville, 1994). These mutants were identified
by direct analysis of leaf or seed fatty acid composition of individual
mutagenized plants by GC (Browse et al., 1986a). One of them,
act1, is deficient in the activity of chloroplast GPAT, the
first enzyme of the prokaryotic pathway, and its leaf fatty acid
composition is characterized by greatly reduced amounts of 16:3 because
the act1 mutation substantially blocks the flux of carbon
into the prokaryotic pathway (Kunst et al., 1988). In this paper we
report the isolation and characterization of a second class of mutants
with reduced levels of 16:3. These mutants also exhibit reduced carbon
flux into the prokaryotic pathway. However, the mutation does not
appear to affect a step in lipid synthesis but instead may limit the
supply of G3P within the chloroplast.
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MATERIALS AND METHODS |
Plant Material and Growth Conditions
The lines of Arabidopsis thaliana (L.) Heynh. described
here were descended from the Columbia wild type. Mutants were isolated from M2 populations obtained after mutagenesis
with ethyl methanesulfonate (Haughn and Somerville, 1986) by directly
analyzing the fatty acid compositions of small tissue samples by GC
(Browse et al., 1986a). The mutant lines were backcrossed to the wild
type three times before being used in any of the experiments reported
here. Plants were grown on soil in controlled-environment chambers at 22°C under continuous fluorescent illumination (150 µmol quanta m 2 s1). Plants used to
isolate chloroplasts were grown for 11 d under continuous
illumination before being transferred to a growth-type chamber in a
day/night cycle at 22°C for another 12 d. The lighting regime in
the growth chamber was 150 µmol quanta m2
s1 for an 8-h day.
Fatty Acid and Lipid Analysis
The overall fatty acid compositions of leaves and other tissues
were determined by GC after derivatization with 2.5% (v/v) H2SO4 in methanol (Miquel
and Browse, 1992). When small tissue samples were analyzed, the same
procedure was used except that fatty acid methyl esters were extracted
into 150 µL (rather than 1 mL) of hexane. Typically, 75 to 100 µL
of this extract could be recovered and transferred to a microvial for
injection onto the gas chromatograph. Usually, a 1-µL aliquot taken
directly from the sample was sufficient for analysis. Samples of leaf
tissue were killed rapidly by immersion in liquid
N2 and ground under liquid
N2 before being extracted and analyzed as
described previously (Miquel and Browse, 1992).
Lipase Positional Analysis
The fatty acid compositions at the sn-1 and
sn-2 positions of individual lipids were determined by
lipase digestion. After TLC lipids were extracted from the silica gel
by the method of Bligh and Dyer (1959). The protocol for digestion with
Rhizopus sp. lipase, including purification of the
lyso-derivatives and fatty acids, was that described by
Siebertz and Heinz (1977), except that 50 mM
H3BO4 was added to the
buffer used for lipase digestion to minimize intramolecular acyl
transfer on the lyso-lipids produced. Fatty methyl esters
were formed from untreated lipids, lyso-lipids, and fatty
acids as described above, and the fatty acid composition of each
compound was determined by GC.
Chloroplast Isolation
Leaves of the wild type and mutant were harvested at the end of
the night period. Ten grams fresh weight of leaves was homogenized using a Polytron (Kinematica, Lucern, Switzerland; two to three 5-s
bursts) in 125 mL of semifrozen buffer containing 0.35 M
sorbitol, 25 mM Hepes-KOH, pH 7.8, and 2 mg
mL1 defatted BSA. The homogenates were filtered
through two layers of prewetted Miracloth (Calbiochem) and centrifuged
at 2,600g for 1 min in an HB-4 swinging-bucket rotor
(Beckman). Pellets were gently resuspended with a small volume of cold
wash buffer that contained 0.33 M sorbitol, 10 mM Hepes-KOH, pH 7.8, and 2.5 mM EDTA. This
plastid suspension was layered on top of a preformed Percoll
(Pharmacia) gradient. The gradient was self-generated by mixing 16 mL
of 100% Percoll in 0.33 M sorbitol and 16 mL of wash
buffer, and centrifuging for 20 min at 27,000g (SS34 rotor, Beckman). The chloroplasts were purified by centrifuging the gradient at 14,000g (HB-4 rotor) for 1 min. The intact chloroplasts
(the green band deeper in the gradient) were collected, diluted at least three times with wash buffer, and then centrifuged at
2,600g for 1 min as described previously. Finally, the
intact chloroplasts were suspended in wash buffer and used immediately.
All operations were carried out at 4°C.
Enzyme Assay
The PAPase was assayed within isolated envelope membranes,
according to assay 2 of Malherbe et al. (1992). Radioactive PA was
synthesized in situ as purified envelope membranes were loaded with PA
synthesized by acylation of
sn-[14C]G3P (specific radioactivity,
5.7 × 1012 Bq/mol; NEN Life Sciences
Products). PAPase activity was then measured by following PA conversion
into DAG. Typically, the reaction mixture contained 100 to 150 µg of
fatty acids from envelope membranes and 2 to 3.5 mg from stromal
proteins.
Exogenous Glycerol Treatment
Wild-type and mutant plants grown under continuous illumination as
described above were 14 d old at the start of the treatment. Glycerol solutions in double-distilled water (10 or 50 mM)
were sprayed on plants at different times during a period of 80 h
using a perfume atomizer. Control plants were sprayed with water. The volumes used were 500 µL for each treatment of 25 plants during the
first 12 h, 650 µL for treatments from 24 to 36 h, and 800 µL after 48 h until the end of the experiment. The surface of the leaves was dry within 30 min after spraying. Thirty minutes after
the 36- and 80-h treatments, 6 to 15 plants from each treatment and
from each genotype were harvested and weighed individually before being
frozen in liquid N2. Total lipids were extracted from each sample and analyzed as described above.
Other Assays
Chloroplast integrity was determined as described by Heber and
Santarius (1970). Protein concentration was determined by the method of
Bradford (1976) using BSA as a standard.
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RESULTS |
Genetic Analysis
The mutant lines JB19 and EMS 5 no. 1 were isolated without
selection by screening M2 progeny of ethyl
methanesulfonate-mutagenized seeds for altered leaf fatty acid
composition. The mutant lines were identified as being deficient in
16:3. Genetic complementation tests indicated that the two lines have a
lesion at the same locus (data not shown). Therefore, we characterized
only one of the lines in detail. The representative mutant line JB19
was normal in appearance and growth characteristics but could be
readily distinguished from the wild type by the reduced amount of 16:3 in its leaf lipids (Table I). The mode of
inheritance of this altered fatty acid composition was determined by
reciprocal crosses between line JB19 plants and wild-type Arabidopsis.
Leaves of the F1 progeny showed a slightly
decreased amount of 16:3 compared with the wild type (Table I),
suggesting that the wild-type allele is incompletely dominant. The
frequency of individuals with the mutant phenotype in the
F2 population resulting from self-fertilization of F1 plants was also measured by GC of leaf
samples. Of 96 F2 plants analyzed, 21 had fatty
acid compositions similar to those of plants of the original JB19 line,
whereas the remaining individuals had leaf fatty acid compositions
similar to those of plants of the wild type or the
F1 hybrid. This pattern of segregation is a good
fit ( 2 = 0.374, P > 0.6) to the 3:1
hypothesis and indicates that the altered fatty acid composition is
caused by a single nuclear mutation at a locus we have designated
gly1. Consequently, the two lines JB19 and EMS 5 no. 1 were
designated gly1-1 and gly1-2, respectively.
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Table I.
Fatty acid composition of total leaf lipids from
wild-type (WT) and gly1-1 mutant Arabidopsis
Homozygous (gly1-1) and heterozygous (F1 of the
cross WT × gly1-1) mutant plants were grown together
with the wild type. Results are means ± SE,
n = 24.
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An Arabidopsis mutant, act1, with greatly reduced 16:3
amounts in its leaf lipids (1-2% of total fatty acids) has previously been characterized as being deficient in chloroplast GPAT (Kunst et
al., 1988). Reciprocal crosses between gly1-1 and
act1 produced F1 progeny, the fatty
acid composition of which was similar to that of the wild type. This
genetic complementation indicates that gly1-1 and
act1 are not allelic and that gly1-1 is not
deficient in chloroplast GPAT activity.
Biochemical Characterization
In mutant leaves the nearly 3-fold reduction in 16:3 was not
accompanied by any increase in the precursors 16:0, 16:1, or 16:2, but
was compensated for by increased 18:1, 18:2, and 18:3 amounts (Table
I). This lack of precursor accumulation indicates that the mutation in
gly1-1 is not attributable to a reduction in desaturation of
16-carbon fatty acids. In plant roots, which contain a predominance of
extrachloroplastic membranes, and in seeds, which contain large amounts
of triglycerides, the prokaryotic pathway does not contribute
significantly to lipid synthesis. Comparison of the overall fatty acid
composition of the roots and mature seeds from the mutant and the wild
type showed no detectable difference. Although we have shown that the
two mutations act1 and gly1-1 are not allelic
(see above), the comparison between their respective overall leaf fatty
acid composition suggests that the mutation in gly1-1 likely
affects the flux of fatty acids into the prokaryotic pathway. Finally,
because 16:3 is synthesized exclusively in chloroplasts by sequential
desaturation of 16:0 acyl groups of galactolipids (Roughan et al.,
1979; Roughan and Slack, 1982), the remaining amount of 16:3 in
gly1-1 leaf lipids suggests that the mutation affects a
biochemical step that is partially redundant, or that the mutation
incompletely blocks a step in the prokaryotic pathway.
The biochemical consequences of the gly1-1 mutation are
shown more clearly by an analysis of individual lipids extracted from leaf tissue of wild-type and mutant plants (Table
II). The data indicated that the changes
in the proportions of the various polar lipids in the mutant affected
only the chloroplast lipids MGD, DGD, SL, and PG. In gly1-1
the mole fraction of MGD decreased by 10% compared with that in the
wild type, whereas DGD increased by 19%. However, when chloroplast
lipids were considered as a whole, there was no difference between
gly1-1 and the wild type. The differences in the fatty acid
compositions of individual lipids were more informative regarding the
nature of the mutation. In MGD, DGD, and SL the reduction in 16-carbon
fatty acid amounts was compensated for by higher amounts of 18-carbon
fatty acids, as indicated by the ratio C-18/C-16 fatty acids (Table
II). This ratio increased by 1.5-, 1.8-, and 2.5-fold for SL, DGD, and
MGD, respectively, indicating a reduced synthesis of prokaryotic-type molecules for these lipids. The data on the lipid composition and on
the fatty acid compositions of individual lipids showed that the
reduced synthesis of galactolipids and SL by the prokaryotic pathway in
the mutant was entirely compensated for by increased production of
these lipids via the eukaryotic pathway.
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Table II.
Fatty acid composition of leaf lipids from
wild-type (WT) and gly1-1 mutant Arabidopsis
Values represent the averages of three samples.
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In wild-type Arabidopsis the prokaryotic pathway is responsible for
producing approximately 70% of the total leaf MGD, 12% of the DGD,
63% of the SL, and 85% of the PG, as indicated by the amounts of
16-carbon fatty acids at the sn-2 position of the glycerol
(Browse et al., 1986b). To quantitate the effect of the mutation on the
flux of fatty acids through the prokaryotic pathway, purified lipids
were digested with Rhizopus sp. lipase and
the fatty acid compositions of the lyso-derivatives and
released fatty acids were determined (Table
III). This analysis indicated that in the
mutant, only 34% of the MGD, 24% of the DGD, and 39% of the SL were
synthesized through the prokaryotic pathway. By contrast, the synthesis
of PG was not affected. The other polar lipids contained >90%
18-carbon fatty acids at the sn-2 position of the glycerol,
indicating that they were produced by the eukaryotic pathway.
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Table III.
Mass and fatty acid compositions of wild-type (WT)
and gly1-1 mutant Arabidopsis leaf lipids and their lyso-derivatives
Polar lipids were separated by two-dimensional TLC. The mass and fatty
acid compositions of the lipids and their lyso-derivatives, resulting from digestion with Rhizopus sp. lipase, were
determined by GC analysis as outlined in ``Materials and Methods''.
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A previous detailed analysis of wild-type Arabidopsis showed that for
every 1000 fatty acid molecules made in the chloroplast, 615 enter the
eukaryotic pathway (117 as 16-carbon fatty acids and 498 as 18-carbon
fatty acids). A similar analysis of gly1-1 indicated a 29%
increase in flux through the eukaryotic pathway, which was made up of
85% 18-carbon fatty acid chains (Fig.
1). However, the C-18/C-16 ratio in PC,
PE, and PI is the same as in the corresponding lipids of the wild type
(Table II). In contrast, the C-18/C-16 ratio in the galactolipids and
SL of the mutant in each case is more than the ratio calculated for
these lipids synthesized by the eukaryotic pathway in the wild type
(table IV of Browse et al., 1986b). Thus, the additional 18-carbon
fatty acids entering the eukaryotic pathway in the mutant are found specifically in the additional quantities of chloroplast lipids (galactolipids and SL) that are produced by the eukaryotic pathway in
response to the loss of the prokaryotic pathway. This situation is also
characteristic of the act1 mutant, in which the loss of the
prokaryotic pathway is almost complete except for the synthesis of PG.
In act1 there is a 50% increase of the flux of fatty acids through the eukaryotic pathway that is made up of 86% 18-carbon fatty
acids (Kunst et al., 1988).
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| Figure 1.
Flow diagram of fatty acid fluxes (mol/1000 mol)
during lipid synthesis by wild-type (WT) and gly1-1
mutant Arabidopsis leaves. NFA, Nonesterified fatty acids.
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Another analogy with the act1 mutant is that the mutation
gly1-1 causes a significant increase in the amount of 18:1
and a decrease in the amount of 18:3 in all of the extrachloroplastic lipids. In these lipids there is only a slight effect on the amount of
18:2 (Table II). The increase in 18:1 amounts in these lipids in the
mutant relative to the wild type reflects a 5 to 7% reduction in the
extent of 18:1 desaturation, suggesting that the ER 18:1 desaturase may
be unable to completely metabolize the increased flux of lipid through
the eukaryotic pathway in the mutant.
PAPase Activity
Because the synthesis of PG by the prokaryotic pathway was not
affected in the mutant, we first considered the possibility that
gly1-1 plants were deficient in the enzyme PAPase. The
chloroplast PAPase provides the DAG moieties (Joyard and Douce, 1977)
used for the synthesis of the prokaryotic molecular species of MGD, DGD, and SL that characterize 16:3 plants (Browse and Somerville, 1991). In the case of 18:3 plants, the chloroplast PAPase is not functional and the chloroplast lipids are synthesized from eukaryotic DAG molecular species (Browse and Somerville, 1991). Therefore, the
gly1-1 phenotype could be explained by a mutation that
reduced (but did not eliminate) PAPase activity. We assayed the
activity of the chloroplast PAPase (Malherbe et al., 1992) and found
that the activity is increased in the mutant compared with wild-type controls (Fig. 2). These results indicate
that PAPase is not decreased in the mutant.
G3P Supply for Lipid Biosynthesis in Chloroplasts
Another hypothesis that could account for the phenotype of
gly1-1 plants is a defect in the availability of chloroplast
G3P, which provides the glycerol backbone of the lipids synthesized by
the prokaryotic pathway (Frentzen, 1993). Experiments with isolated
spinach chloroplasts and intact leaf tissues indicate that flux of
fatty acids into the prokaryotic pathway is sensitive to changes in G3P
availability. Roughan et al. (1980) showed that chloroplasts incubated
in a basal medium contained 23% of the incorporated label in
glycerolipids (representing the prokaryotic pathway), whereas more than
70% was incorporated into unesterified fatty acids and acyl-CoAs,
which are precursors for the eukaryotic pathway. Addition of 0.48 mM G3P to the basal medium increased glycerolipid products
to 49% of the total radioactivity and decreased unesterified fatty
acids plus acyl-CoAs to 40%. When glycerol was supplied to leaves of
spinach plants, it increased the size of the G3P pool and increased the
flux of [14C]acetate label into prokaryotic
lipids (Gardiner et al., 1982). These results suggest that if the
gly1-1 mutation is a lesion reducing the availability of G3P
in the chloroplast, then exogenous application of glycerol should
result in chemical complementation of the mutant phenotype.
Because 16:3 is produced only by the prokaryotic pathway, it was
possible to use this fatty acid to monitor the effects of glycerol
treatments. A typical experiment in which glycerol was applied to
wild-type and gly1-1 plants is described in Figure 3. The control plants in these
experiments received an application of water instead of glycerol
solutions. In wild-type plants glycerol caused a small but consistent
increase in 16:3 in the total leaf lipids. By contrast, after 80 h
of treatment with 50 mM glycerol, gly1-1 plants
exhibited a 2-fold increase in the proportion of 16:3 in total lipids
to levels that were similar to those of untreated wild-type plants.
When the individual lipids were purified and their fatty acid
composition analyzed, we determined that the level of 16:3 in MGD of
gly1-1 plants after 80 h of 50 mM glycerol application had doubled to 29% compared with that of the control gly1-1 plants. By contrast, in wild-type plants the increase
was only to 3%. Therefore, raising the G3P amounts in
gly1-1 plants led to a notable alleviation of the altered
fatty acid composition. This suggests that the concentration of G3P in
gly1-1 chloroplasts is not sufficient to meet the
requirement for chloroplast lipid synthesis through the prokaryotic
pathway. Application of glycerol is known to inhibit photosynthesis
(Leegood et al., 1988), but both the wild-type and mutant plants
remained healthy throughout the duration of our experiments.
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| Figure 3.
Effect of exogenous glycerol treatment of
wild-type and gly1-1 mutant Arabidopsis on their leaf
fatty acid composition. Wild-type and mutant plants were supplied with
exogenous glycerol by repeatedly spraying rosette leaves with water
containing 0, 10, or 50 mM glycerol at the times indicated.
Thirty minutes after the 36- and 80-h treatments, 6 to 15 plants for
each glycerol treatment and from each genotype were harvested and total
leaf lipids were extracted from each sample. The bar graphs show 16:3
as a percentage of total fatty acids. Striped bars, Wild type; shaded
bars, gly1-1; G, glycerol treatment during the 1st 12 h.
Repeat treatments on subsequent days are indicated by tick marks. H,
Harvest.
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DISCUSSION |
Studies of lipid metabolism pathways using a genetic approach have
revealed the existence of regulatory mechanisms that coordinate the
activity of the two pathways for glycerolipid biosynthesis in higher
plants. For example, the deficiency in activity of chloroplast GPAT in
act1 mutants is compensated for by increased synthesis of
chloroplast glycerolipids via the eukaryotic pathway (Kunst et al.,
1988). This and studies of other Arabidopsis mutants (Browse et al.,
1989; Kunst et al., 1989; Miquel and Browse, 1992) indicate that lipid
metabolism is regulated to ameliorate the consequences of the lesion in
each mutant by altering the flux through the two pathways of
glycerolipid biosynthesis. Mutant gly1-1 plants are no
exception, and the decreased synthesis of prokaryotic species of
chloroplast lipids was entirely compensated for by increased production
of these lipids via the eukaryotic pathway. This reduced synthesis
through the prokaryotic pathway did not result in an increase of
16-carbon chains in the eukaryotic pathway. Instead, the 16:0-ACP was
apparently elongated and desaturated to 18:1-ACP before export from the
chloroplast. The overall C-18/C-16 ratio was then increased from 1.96 in the wild type to 3.5 in the mutant. The C-18/C-16 ratios of
extrachloroplastic lipids were unchanged, suggesting that the amount of
16:0 export was not regulated by the availability of 16:0. Rather, the
increase in the overall ratio indicated that elongation is regulated by
availability of the substrate (16:0) and that this is determined by
competition between alternative pathways of 16-carbon fatty acid
metabolism. The mutation in gly1-1 is not an allele of
act1, the structural gene for GPAT. Direct assays of PAPase
activity in wild-type and mutant plants allowed us to indirectly test
for a lesion in a third enzyme of the prokaryotic pathway, LPAAT.
Assays of the PAPase activity necessitated a prior loading of
chloroplast envelopes with radiolabeled PA and, to this effect,
purified chloroplast envelopes were incubated with stromal proteins and
the appropriate cofactors (Malherbe et al., 1992). The stroma is the
source of the GPAT, which forms lyso-PA, and the envelope
contains the LPAAT, which forms PA (Joyard and Douce, 1977). It has
been shown that the GPAT possesses a specificity for G3P and
exclusively catalyzes the acylation of the sn-1 position of
the acyl acceptor (Frentzen, 1986). From this we inferred that the
presence of radiolabeled PA in the envelopes resulted from the sole
action of the LPAAT on lyso-PA. Thus, the gly1-1
mutation is not a deficiency in LPAAT activity.
The results of the exogenous glycerol treatment and the fact that the
gly1-1 phenotype could not be explained by a deficiency in
the enzymes of the prokaryotic pathway pointed to a possible defect in
the G3P supply for lipid biosynthesis in chloroplasts. The experiments
in which exogenous glycerol provides for chemical complementation of
the gly1-1 phenotype are consistent with a mutant lesion
affecting the supply of G3P within the chloroplast. Because G3P is
present in (at least) the chloroplast and cytoplasm of leaf cells it is
not possible to measure the chloroplast G3P pool in vivo. In plants G3P
can be synthesized via essentially three different reaction sequences
(Frentzen, 1993). It can be formed from DHAP by the action of an
NAD+-G3P oxidoreductase (EC 1.1.1.8 or DHAP
reductase) in both the chloroplast and cytoplasm of leaves from higher
plants (Santora et al., 1979; Gee et al., 1988a, 1988b, 1988c, 1989;
Kirsch et al., 1992). G3P can also be formed by the pathway
DHAP  glyceraldehyde 3-phosphate glyceraldehyde glycerol G3P in the cytoplasm (Ghosh and Sastry, 1988). The last enzyme of the
pathway, glycerol kinase, has been detected in all plant organs
(Hippman and Heinz, 1976; Sadava and Moore, 1987) and especially in
germinating seeds (Huang and Beevers, 1975; Hippman and Heinz, 1976;
Finlayson and Dennis, 1980) and appears to be the rate-limiting enzyme
of this pathway. Except in developing groundnut seeds (Ghosh and
Sastry, 1988), both G3P synthesis pathways are found, therefore raising the question of the respective roles and importance of the different pathways and their regulation. Given the uncertainties surrounding the
source of G3P in the chloroplast it is likely that further studies on
the gly1-1 mutant will help to resolve the questions in this
area of biochemistry.
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FOOTNOTES |
1
This work was supported by the Centre National
de la Recherche Scientifique and the Université Victor Segalen
(Bordeaux, France), the National Science Foundation (grant no.
IBN-9407902), and the Agricultural Research Center, Washington
State University.
*
Corresponding author; e-mail miquel{at}biomemb.u-bordeaux2.fr; fax
33-5-5651-8361.
Received January 29, 1998;
accepted April 7, 1998.
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ABBREVIATIONS |
Abbreviations:
ACP, acyl carrier protein.
DAG, diacylglycerol.
DGD, digalactosyldiacylglycerol.
DHAP, dihydroxyacetone phosphate.
G3P, glycerol-3-phosphate.
GPAT, acyl-ACP:sn-G3P
acyltransferase.
LPAAT, acyl-ACP:sn-1-acylglycerol-3-phosphate acyltransferase.
MGD, monogalactosyldiacylglycerol.
PA, phosphatidic acid.
PAPase, phosphatidate phosphatase.
PC, phosphatidylcholine, PE,
phosphatidylethanolamine.
PG, phosphatidylglycerol.
PI, phosphatidylinositol.
SL, sulfoquinovosyldiacylglycerol (sulfolipid).
X:Y, a fatty acyl group containing X carbon atoms and Y double bonds
(cis unless specified).
16:0, palmitate.
16:1, palmitoleate.
16:2, hexadecadienoic acid.
16:3, hexadecatrienoic acid.
18:0, stearate.
18:1, oleate.
18:2, linoleate.
18:3, linolenate.
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ACKNOWLEDGMENT |
We thank Dr. Jim Tokuhisa for the gift of EMS 5 no. 1.
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Top
Abstract
Introduction
, Wild type; 
, gly1-1.