Mutation of the TGD1 Chloroplast Envelope Protein Affects
Phosphatidate Metabolism in Arabidopsis
Changcheng Xu,aJilian Fan,aJohn E. Froehlich,bKoichiro Awai,aand Christoph Benninga,1
aDepartment of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824
bDepartment of Energy, Plant Research Laboratory, Michigan State University, East Lansing, Michigan, 48824
Phosphatidate (PA) is a central metabolite of lipid metabolism and a signaling molecule in many eukaryotes, including
plants. Mutations in a permease-like protein, TRIGALACTOSYLDIACYLGLYCEROL1 (TGD1), in Arabidopsis thaliana caused
the accumulation of triacylglycerols, oligogalactolipids, and PA. Chloroplast lipids were altered in their fatty acid
composition consistent with an impairment of lipid trafficking from the endoplasmic reticulum (ER) to the chloroplast
and a disruption of thylakoid lipid biosynthesis from ER-derived precursors. The process mediated by TGD1 appears to be
essential as mutation of the protein caused a high incidence of embryo abortion. Isolated tgd1 mutant chloroplasts showed
a decreased ability to incorporate PA into galactolipids. The TGD1 protein was localized to the inner chloroplast envelope
and appears to be a component of a lipid transporter. As even partial disruption of TGD1 function has drastic consequences
on central lipid metabolism, the tgd1 mutant provides a tool to explore regulatory mechanisms governing lipid homeostasis
and lipid trafficking in plants.
Chloroplasts harbor one of the most complex membrane sys-
tems (thylakoids) in nature to conduct photosynthesis. Efforts in
gaining a fundamental understanding of chloroplast biogenesis
have to address questions of membrane assembly, including
polar lipid biosynthesis and lipid trafficking. The predominant
thylakoid membrane lipids are the galactolipids (Do ¨rmann and
Benning, 2002), which are assembled in the model plant Arabi-
with the chloroplast envelope membranes (Benning and Ohta,
2005). A basic model of galactolipid biosynthesis in Arabidopsis
is shown in Figure 1. In the wild type, the bulk of galactolipids is
synthesized by monogalactosyldiacylglycerol (MGDG) synthase
1 (MGD1) (Shimojima et al., 1997; Jarvis et al., 2000) and
digalactosyldiacylglycerol (DGDG) synthase 1 (DGD1) (Do ¨rmann
et al., 1999). Interestingly, MGD1 is associated with the inner
2001) and DGD1 with the outer envelope (Froehlich et al., 2001),
raising mechanistic questions of MGDG movement from the
inner to the outer envelope and of DGDG transfer back from the
outer to the inner envelope (Figure 1). The specific biological
function or the evolutionary advantages, if any, of the arrange-
ment of the galactoglycerolipid biosynthetic machinery are not
well understood. To complicate matters, two parallel pathways
contribute to chloroplast lipid biosynthesis in many plants (e.g.,
Arabidopsis, Figure 1) as originally proposed by Roughan and
Slack (Roughan et al., 1980; Roughan and Slack, 1982). The
eukaryotic pathway for thylakoid lipid biosynthesis encom-
passes the export of fatty acids synthesized in the chloroplast,
their incorporation into phosphatidates (PAs), and, subse-
quently, other phospholipids at the endoplasmic reticulum
(ER). The biosynthesis of thylakoid lipids by the eukaryotic
pathway necessitates the return of either PA directly and/or the
return of phosphatidylcholine (PC) to the plastid, which would
require the conversion of PC to PA by a phospholipase D at the
outer chloroplast envelope and, subsequently, the dephosphor-
ylation of PA by a PA phosphatase (PAP) to diacylglycerol
(DAG), which is the precursor of MGDG biosynthesis at the
inner chloroplast envelope. Alternatively, fatty acids are di-
rectly incorporated at the plastid inner envelope into PA, which
enters MGDG biosynthesis as described for the eukaryotic
pathway. This de novo assembly of MGDG in the plastid rep-
resents the prokaryotic pathway. According to this hypothe-
sis, a PAP activity plays a central role in both pathways (Figure
1). While PAPs are known for Arabidopsis and other plants
(Pearce and Slabas, 1998; Pierrugues et al., 2001), a spe-
cific isoform(s) involved in the eukaryotic or prokaryotic path-
ways has not yet been identified. Distinct molecular species
of MGDG or any other thylakoid lipid arise from the two
pathways due to the different substrate specificities of the
lysophosphatidic acid acyltransferases associated with the ER
or the inner chloroplast envelope, respectively, and the fatty
acid composition of different MGDG molecules are diagnostic
for their origin (Heinz and Roughan, 1983). While the contribu-
tions of the eukaryotic and prokaryotic pathways can be greatly
different in different plant and algal species (Heinz, 1977;
Mongrand et al., 1998), they are nearly equal in Arabidopsis
(Browse et al., 1986).
1To whom correspondence should be addressed. E-mail benning@msu.
edu; fax 517-353-9334.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Christoph Benning
WOnline version contains Web-only data.
Article, publication date, and citation information can be found at
The Plant Cell, Vol. 17, 3094–3110, November 2005, www.plantcell.org ª 2005 American Society of Plant Biologists
A central metabolite of the eukaryotic and prokaryotic path-
ways of thylakoid lipid assembly is PA. In wild-type Arabidopsis,
it represents only a minor fraction of the polar lipids. In fact, PA
released from phospholipids by specific lipases acts as a signal-
ing molecule in a variety of pathways in plants, and PA levels are
under strict control (Lee et al., 2003; Potocky et al., 2003; Zhang
et al., 2003; Li et al., 2004; Park et al., 2004; Thiery et al., 2004).
It should also be noted that PA is a regulator of phospho-
lipid metabolism in yeast (Loewen et al., 2004) and likely also in
Three trigalactosyldiacylglycerol1 (tgd1) mutant alleles of Arab-
idopsis were identified, and the locus (At1g19800) was named
based on the unusual accumulation of oligogalactolipids in the
mutants (Xu et al., 2003). As described here, we discovered that
the tgd1-1 mutant representative for all alleles also accumulates
triacylglycerols (TAGs) and PA. The oligogalactolipids found in
tgd1-1 differed with regard to the glycosidic linkage of the head
group sugars from the typical MGDGs and DGDGs of thylakoid
membranes, suggesting that a processive galactosyl transferase
different from the primary galactolipid biosynthetic enzymes was
responsible. In addition, molecular species of galactolipids de-
rived from the ER pathway of thylakoid lipid biosynthesis were
underrepresented in tgd1-1 such that most of the galactolipid
molecules associated with chloroplast membranes were derived
mutants was predicted to encode a six-membrane-spanning
domain protein with similarity to the permease component of
multipartite bacterial ABC transporters. The tgd1 mutant alleles
were originally isolated in the genetic background of the
digalactolipid-deficient dgd1 mutant (Do ¨rmann et al., 1995).
However, the in-depth analysis of the complex tgd1 mutant
phenotype described here was conducted in the wild-type
background following extensive backcrossing. Extensive analy-
sis revealed subtle morphological phenotypes and a high in-
cidence of embryo lethality due to compromised TGD1 function.
Based on the cumulated results derived from the analysis of
the complex tgd1-1 phenotype and TGD1, it is suggested that
velope involved in the eukaryotic pathway of thylakoid lipid bio-
Impairment of TGD1 Affects Growth and Development
While the tgd1 mutants were originally isolated in the dgd1
mutant background during a suppressor screen (Xu et al., 2003),
it became quickly clear following crosses to the wild type that
mutations in the TGD1 gene cause phenotypes independent of
dgd1, permitting a more direct analysis of TGD1 function in the
wild-type background. Because all three available point mutant
alleles showed comparable biochemical and growth pheno-
primarily on the tgd1-1 allele in this study. All three alleles were
leaky, with tgd1-1 being the most severe with regard to the
developmental phenotypes described here. The growth of the
tgd1-1 mutant line was slightly reduced, and leaves and siliques
could be distinguished from the wild type based on their
stoutness, as summarized in Figure 2. The reduction in growth
was not only visible for green tissues but extended to nongreen
tissues, such as roots, as well (Figure 2C).
Because no knockout allele was readily available, we used
an RNA interference (RNAi) approach to enhance the mutant
phenotype of tgd1-1 and thereby demonstrate the leakiness of
the tgd1-1 allele. Two representative RNAi lines are shown in
lines (Figure 3A), resulting in further diminished growth (Fig-
During the course of handling the tgd1 mutant lines, it was
noted that seed batches reproducibly contained large numbers
of sterile seeds oremptyseed hulls. Inhomozygous tgd1-1 lines,
nearly 50% of the seeds were aborted (Figures 4A and 4B),
a phenotype that was already visible for immature seeds of
opened siliques. The ratio of aborted seeds approached 90%
(409 out of 481 total for RNAi line-1 and 519 out of 583 total for
RNAi line-2; Figure 3C) for the tgd1-1/TGD1-RNAi lines. This
seed phenotype, as all others studied for tgd1-1, was rescued
by ectopic expression of the TGD1 wild-type cDNA under the
control of the cauliflower mosaic virus (CaMV) 35S promoter or
by a cosmid that harbored a genomic fragment containing the
wild-type gene with its endogenous gene promoter (Figure 4B).
Approximately 50% of the seeds of the tgd1-1 mutant were
aborted (Figure 4B). When embryos forming in siliques of
the tgd1-1 mutant line were directly examined from 2 to 6 d
after flowering, many were found arrested at the heart stage
(Mansfield and Briarty, 1991) just before the greening of the
plastid was expected to begin (Figure 4C).
Figure 1. Two-Pathway Scheme for Galactolipid Biosynthesis.
Lipid flow is shown with solid arrows. Membranes shown in gray are as
follows: iE, inner chloroplast envelope; oE, outer chloroplast envelope;
Thy, thylakoids. Proteins (round shapes) shown are: ACT1, plastidic
glycerol-3-phosphate acyltransferase; DGD1, major DGDG synthase;
MGD1, major MGDG synthase; PAP, plastidic PAP. Lipids are encircled,
and molecular species derived from the eukaryotic pathway carry the
subscript ‘‘E,’’ those derived from the prokaryotic pathway ‘‘P,’’ and
mixtures derived from both pathways ‘‘E/P.’’
Altered Phosphatidate Metabolism in tgd1 3095
The Eukaryotic Pathway Is Predominant in
Based on the previous biochemical analysis of the tgd1 mutants
(Xu et al., 2003), it was concluded that the mutation in tgd1-1
causes an impairment of the eukaryotic (ER) pathway for
thylakoid lipid biosynthesis (cf. Figure 1). To corroborate this
hypothesis by genetic means, we crossed the homozygous
tgd1-1 mutant with a homozygous act1 mutant. The act1 mutant
is deficient in the plastidic glycerol 3-phosphate acyltransferase
(Figure 1) blocking the prokaryotic pathway of galactolipid bio-
synthesis. Its chloroplast galactolipids are derived from the
eukaryotic pathway (Kunst et al., 1988). Given the deficiency of
the eukaryotic pathway in the tgd1-1 mutant mentioned above,
the tgd1-1 act1 double mutant was expected to be unable to
are essential components of all plastid membranes. Indeed, no
viable homozygous tgd1-1 act1 double mutants were recovered
from the F2 seeds. Screening 98 F2 plants for the diagnostic loss
of 16:3 carbon fatty acids, 17 homozygous act1 mutants were
identified. Genotyping these atthe TGD1locus, 10were foundto
beheterozygous fortgd1-1 and 7were homozygouswild-type at
the TGD1 locus. When developing siliques of the F1 plants from
the cross between homozygous tgd1-1 and act1 mutants were
opened (Figure 3C), three classes of seeds or seed-like struc-
tures were discernible: green normal-looking seeds, white seeds
of the same size as the wild type, and small white seed-like
of nearly 1/16 (39 out of 602, 6.45%) which is the expected
frequency of double homozygous mutant seeds in this F2
population. Furthermore, when F3 seeds in siliques of F2 lines
homozygous for act1 and heterozygous for tgd1-1 were ana-
lyzed, ;28% (121 out of 432) were very small and white. We
propose that these very small seed-like structures represent the
double homozygous mutant incapable of developing a viable
seed due to their inability to synthesize galactolipids. The white,
large seeds are presumably homozygous for tgd1-1 only.
Because embryos of Arabidopsis develop chloroplasts with
extensive thylakoid membranes at the heart stage (Mansfield
and Briarty, 1991), we investigated the contribution of the pro-
karyotic and eukaryotic pathways to thylakoid lipid biosynthesis
in developing seeds of the tgd1-1 mutant and wild-type lines,
suspecting that the eukaryotic pathway might be more pre-
dominant in seeds than in leaves, as seed metabolism in general
is largely directed toward TAG biosynthesis involving the eu-
karyotic pathway. All seeds (phenotypically mutant or wild-type)
derived from a developing homozygous mutant silique were
combined and extracted for further lipid analysis and compared
Figure 2. Impaired Growth and Development of the tgd1-1 Mutant.
(A) Morphology of 7-week-old wild-type and tgd1-1 plants grown on soil.
(B) Comparison of leaves (left) and siliques (right) of the wild type and the
tgd1-1 mutant. Bar (right panel only) ¼ 1 mm.
(C) Growth rates for shoots (aerial parts; top) and roots (bottom) of wild-
type and tgd1-1 mutant plants grown on agar-solidified Murashige and
Skoog (MS) medium with 1% sucrose. For each point, 15 plants for shoot
and 10 plants for root measurements were averaged. Standard errors are
indicated. Squares represent the wild type, and circles represent the
3096The Plant Cell
with seed extracts of developing wild-type siliques of compara-
ble age. As shown in Figure 4D, the polar lipid composition of
mutant seeds was biased toward a decrease of chloroplast
lipids, such as the two galactolipids and an increase of ER lipids,
such as PC, consistent with the arrest of development of mutant
embryos at the stage of chloroplast formation. Interestingly, the
relative amount of phosphatidylethanolamine, unlike PC, a lipid
the mutant. Possibly, PC is more dominant in the ER of embryos
than phosphatidylethanolamine because of its precursor role in
oil biosynthesis (Ohlrogge and Browse, 1995). Focusing on the
exclusive chloroplast lipid MGDG, we observed that 18-carbon
fatty acids were predominant in the wild-type MGDG but re-
ducedin mutantMGDG(Figure 4E).Ingeneral, molecular MGDG
species containing two 18-carbon fatty acids in their DAG
backbone are thought to be derived from the eukaryotic path-
way, while molecular species of MGDG with an 18-carbon fatty
acid in the first position of DAG and a 16-carbon fatty acid in the
second positionarederived fromthe prokaryotic pathway (Heinz
and Roughan, 1983). Given the predominance of 18-carbon fatty
acids in MGDG of the wild type, it was apparent that the eu-
karyotic pathway dominates in developing Arabidopsis seeds
while it contributes 50% of thylakoid lipids in leaves (Browse
et al., 1986). As all results on the lipid metabolism in the tgd1
mutant point to a general defect in the eukaryotic pathway of
thylakoid lipid biosynthesis, the tgd1-1 mutation should have
severe consequences for the ability of the embryo to generate
sufficient lipids for plastid membrane biogenesis. Therefore, the
observed predominance of the eukaryotic pathway in wild-type
seeds explains the high incidence of seed abortion in the leaky
tgd1-1 mutant and the strongly increased incidence of seed
abortion in the tgd1-1/TGD1 RNAi lines.
Oligogalactolipids, TAG, and PA Accumulate in
The tgd1 mutants were originally identified by their accumulation
erol (TGDG). A comparison of polar lipids in the tgd1-1 mutant
and the wild type is shown in Table 1. While TGDG was not
detectable in the wild type, it was present at ;1.4 mol % in the
slightly reduced and that of DGDG was slightly more (by 29%). In
the wild type, DGDG is mostly of eukaryotic origin presumably
due to the location of the respective DGD1 glycosyltransferase
on the outside of the outer envelope or the substrate specificity
of this enzyme. Therefore, the short supply of molecular lipid
species from the eukaryotic pathway in the tgd1-1 mutant might
explain this decrease in DGDG. The relative amounts of phos-
pholipids overall increased in the mutant. The structural identity
of purified TGDG had been previously determined by mass
spectrometry and nuclear magnetic resonance spectroscopy
(Xu et al., 2003).
During the ultrastructural comparison of wild-type and mutant
cells, we observed peculiar osmium-dense bodies in the cyto-
plasm exclusively in the mutant as shown in Figure 5. Typically,
tight packing of alkane chains associated with membrane lipids
or oil droplets causes an enhancement of osmium staining, and
wehypothesized thatthesebodies mightbelipid droplets. When
lipid extracts from leaves and roots of the mutant were analyzed
for neutral lipids, a lipid cochromatographing with soy seed oil
TAGs was detected (Figure 6A). Digestion of this compound
isolated from mutant leaves and of soy oil TAGs with pancreatic
lipase led to the formation of free fatty acids, diacylglycerol, and
Figure 3. Enhancement of the Morphological Phenotype of tgd1-1 in
TGD1-RNAi Lines and a tgd1-1 act1 Double Mutant.
(A) Reduced amounts of tgd1-1 mRNA in two representative indepen-
dent TGD1 RNAi lines constructed in the homozygous tgd1-1 mutant
background. UBIQUITIN10 mRNA (UBQ10) was used as a control for the
(B) Morphology of 6-week-old plants, as labeled, grown on soil.
(C) Representative siliques 7 d after flowering opened up to expose
the developing seeds. Only in the tgd1 act1 F1 silique are three seed
types visible: wild type, green; mutant, white but same size as the wild
type; mutant, small and white (arrow). Seed ratios are provided in the
text. Bars ¼ 1 mm.
Altered Phosphatidate Metabolism in tgd13097
thereby confirming the identity of the compound accumulating
in the mutant as TAG (see Supplemental Figure 1 online). Soy
seed oil was chosen here as a standard because its fatty acid
composition, unlike that of seed oil from Arabidopsis, resembles
that of Arabidopsis leaf lipids. Indeed, analysis of fatty acid
methylesters derived from the TAGs accumulating in leaves of
the tgd1-1 mutant confirmed the presence of fatty acids typical
for leaves (Figure 7A). Furthermore, the fatty acid composition
was similar to that of PC (Figure 7B), a lipid derived from the
eukaryotic pathway. It is interesting to note that a small amount
of 16:3 fatty acids is present in PC as well as in PA in the tgd1-1
is normally indicative of molecular species derived from the
cochromatographingin both samples,
prokaryotic pathway, and its presence in PC indicates a small
flux of prokaryotic lipid species to the extraplastidic membranes
in the tgd1-1 mutant but not the wild type. To show that the TAG
was not associated with the plastids, chloroplasts were isolated
and analyzed as well. No TAGs were detectable in isolated
chloroplasts (Figure 6A). Taking these data together, it is pro-
posed that leaf cells of the tgd1-1 mutant accumulate TAGs in
droplets in the cytoplasm.
To examine whether central phospholipid metabolism was af-
fected in the tgd1-1 mutant, leaves were labeled with [32P]ortho-
phosphate to detect changes in phospholipid intermediate
pools in the mutant. Two-dimensional thin-layer chromatograms
compound comigrating with an authentic PA standard was
Figure 4. Seed Phenotypes of the tgd1-1 Mutant.
(A) Representative siliques 7 d after flowering opened up to expose the developing seeds. Top, the wild type; center, the tgd1-1 mutant; bottom,
transgenic tgd1-1 mutant complemented by expression of the TGD1 wild-type sense cDNA under the control of the CaMV 35S promoter. Bars ¼ 1 mm.
(B) Frequency of aborted seeds (from left) in the wild type, the tgd1-1 mutant, the tgd1 mutant complemented with the cDNA as described in (A), and the
mutant complemented with a genomic cosmid expressing the wild-type TGD1 gene under the control of the endogenous promoter. Open bars, filled
seed; closed bars, empty seeds. At least 10 siliques per line were averaged. Standard errors are indicated.
(C) Nomarski microscopy of developing seeds of the wild type (top panels) and the tgd1-1 mutant (bottom panels) 2, 4, and 6 d after flowering (DAF).
Bars ¼ 80 mm.
(D) Relative polar lipid content of wild-type and mutant developing seeds. Seeds were analyzed;6 d after flowering. Data from three independent lipid
preparations were averaged. The standard error is indicated. Open bars, wild type; closed bars, tgd1-1 mutant seeds. PE, phosphatidylethanolamine;
PG, phosphatidylglycerol; PI, phosphatidylinositol; SQDG, sulfoquinovosyldiacylglycerol.
(E) Fatty acid composition of the prevalent chloroplast lipid, MGDG. Fatty acids are listed according to the carbon number followed by the number of
double bonds. Data from three independent lipid preparations were averaged. The standard error is indicated. Open bars, wild type; closed bars, tgd1-1
3098The Plant Cell
increased. Determining the relative mole fraction of fatty acid
methylesters of PA over all fatty acids in the sample (wild type,
0.47 6 0.12 mol %; tgd1-1, 2.15 6 0.55 mol %; n ¼ 3, 6SD)
indicated an approximate fivefold increase of relative PA
amounts in the tgd1-1 mutant. The fatty acid composition of
this PA was similar to that of PC (Figures 7B and 7C). Moreover,
positional analysis of fatty acids in PA revealed an enrichment of
18-carbon fatty acids in the sn-2 position of the glycerol
backbone (92.5 6 5 mol %, n ¼ 3, 6SD) consistent with its origin
from the eukaryotic pathway (Heinz and Roughan, 1983). When
intact chloroplasts were isolated from the tgd1-1 mutant, no PA
was detected (data not shown), suggesting that PA found in total
leaf lipid extracts of the mutant is accumulating in extraplastidic
membranes. However, we cannot rule out that any PA present in
the plastids was metabolized during chloroplast isolation.
Impaired Conversion of PA into Galactolipids by
Because eukaryotic PA accumulated in the tgd1-1 mutant and
the eukaryotic pathway of galactoglycerolipid biosynthesis was
generally disrupted, we hypothesized that the transport of PA
and/or conversion of ER-derived PA to the eukaryotic DAG
precursor of the galactolipid biosynthetic machinery (i.e., MGD1)
in chloroplast envelopes was impaired. To test this hypothesis,
chloroplasts were isolated from mutant and wild-type leaves,
and acylcarbon-labeled lipid substrates were tested for their
differential incorporation into MGDG by the two chloroplast
preparations. The results of these experiments are shown in
Figure 8. When DAG, the direct substrate for MGD1, was used,
incorporation into MGDG was slightly increased for mutant
chloroplasts (Figure 8A). The reaction was strictly dependent
on the addition of the second MGD1 substrate, UDP-Gal, but
was independent of phospholipase C, as expected, because
DAG is not a substrate of phospholipase C. However, when
labeled PC was used (Figure 8B), incorporation of label into
MGDG by mutant chloroplasts was generally decreased. The
Phospholipase C generates DAG from PC, and stimulation was
expected. Interestingly, the difference between the mutant and
the wild-type chloroplasts were nearly abolished following phos-
pholipase C treatment. A similar result as for PC was observed
for PA (Figures 8C and 8D). Incorporation of PA into MGDG was
nearly linear for both chloroplast preparations between the
interval of 10 to 30 min, and the rate of PA incorporation was
reduced for the mutant chloroplasts on a chlorophyll basis. This
reaction was dependent on UDP-Gal and stimulated by addi-
tions of cytosol, which also led to an increase in the difference
between the mutant and wild-type chloroplasts (Figure 8D).
Addition of ER fractions was not effective. Cytosol and ER
not yet identified might be needed for the transfer of PA from
liposomes to the plastid envelopes.
Because it was possible that the reduced incorporation of PA
into MGDG by isolated tgd1-1 chloroplasts is caused by a
decreased activity of PAP (Figure 1), we tested the activity of
this enzyme in the two chloroplast preparations. As shown in
Supplemental Figure 2 online, PAP activity was similar in both
preparations and can therefore not account for the observed
decrease in the incorporation of PA into MGDG by isolated
mutant chloroplasts (Figures 8C and 8D). Together with the
documented accumulation of PA in the tgd1-1 mutant (Figure
6B), these results suggest an impairment of the eukaryotic
pathway at the level of PA and its conversion into DAG, the
direct substrate of MGDG biosynthesis.
MGD1 Activity Associated with the Inner Envelope Is
Increased in tgd1-1
To correctly interpret the observed rates of lipid incorporation
into MGDG described above (Figure 8), it was important to know
Table 1. Polar Lipid Composition in Leaves of the Wild Type and the
Lipid Wild Type tgd1-1
43.0 6 1.8
10.4 6 0.1
18.2 6 0.4
2.3 6 0.2
1.2 6 0.1
10.0 6 0.6
15.1 6 1.4
39.6 6 0.3
12.6 6 0.1
12.9 6 0.5
3.4 6 0.7
1.7 6 0.2
11.0 6 0.4
16.8 6 1.2
1.4 6 0.2
PA could not be separated by linear TLC used for this experiment and
had to be analyzed by two-dimensional TLC. Values represent mol % 6
SE of fatty acid methylesters derived from individual lipids. n.d., not
detected. PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI,
phosphatidylinositol; SQDG, sulfoquinovosyldiacylglycerol.
Figure 5. Electron Micrographs of Representative Cells of the Wild Type
and the tgd1-1 Mutant.
Plastoglobuli (pg) in plastids and oil droplets (od) in the mutant cytosol are
indicated by arrows. Top, wild type; bottom, tgd1-1 mutant. Bars ¼ 1 mm.
Altered Phosphatidate Metabolism in tgd13099
whether the activity of MGDG synthase, in particular that of
MGD1, which is the enzyme responsible for bulk MGDG bio-
synthesis in the wild type, was altered in the mutant. The activity
of MGDGsynthasewas assayed directlyusinglabeled UDP-Gal.
No lipids were added to the mixture requiring the utilization of
endogenous DAG generated in the envelope membranes. As
shown in Figure 9A, under these conditions, the rate of UDP-Gal
incorporation into MGDG was increased approximately fivefold
in isolated mutant chloroplasts. To corroborate that this activity
was due to MGD1 and not to other described galactosyltransfer-
ase activities, chloroplasts were treated with either thermolysin or
Figure 6. Accumulation of TAGs and PA in the tgd1-1 Mutant.
(A) TLC of lipid extracts of wild-type and tgd1-1 tissues and isolated
chloroplasts (Chl) as indicated. A neutral lipid TLC system was used as
described in Methods, and lipids were visualized by iodine staining. O,
(B) Two-dimensional chromatogram of phospholipids from leaves of the
wild type (top) and the tgd1-1 mutant (bottom). Lipids were32P-labeled
and visualized by autoradiography. PE, phosphatidylethanolamine; PG,
phosphatidylglycerol; PI, phosphatidylinositol.
Figure 7. Fatty Acid Composition of Relevant Lipids.
(A) Fatty acid (FA) composition of TAG accumulating in the tgd1-1
mutant in root (hatched bars) or leaf (closed bars) tissues.
(B) Fatty acid composition of PC isolated from leaves of the wild type
(open bars) and the tgd1-1 mutant (closed bars).
(C) Fatty acid composition of PA isolated from leaves of the wild type
(open bars) and the tgd1-1 mutant (closed bars).
For data in all three panels, three independent extracts were averaged,
and the standard error is shown. Fatty acids are designated according to
their number of carbons:number of double bonds.
3100 The Plant Cell
buffer only, reisolated, and incubated with substrate. Thermo-
lysin can degrade surface-exposed proteins localized to the outer
envelope membrane, such as processive galactosyl transferase
(Xu et al., 2003), DGD1 (Froehlich et al., 2001), or MGD2 and
MGD3 (Awai et al., 2001), while not penetrating into the in-
termembrane space and degrading proteins, such as MGD1,
which is proposed to be associated with the inner envelope
membrane (Miege et al., 1999; Awai et al., 2001). While the
formation of DGDG (Figure 9A) and TGDG (Figure 9A) was
thermolysin sensitive, the formation of MGDG was not (Figure
9A). This result was consistent with either an activation of MGD1
or greater availability of the endogenous lipid precursor of the
MGD1 reaction in the tgd1-1 mutant. We determined that the
increase of MGDG formation in the mutant was not due to
increased MGD1 gene expression as shown in Figure 9B.
Considering this increase in MGD1 activity, the result for the
decrease of PA incorporation into MGDG by the mutant chloro-
plasts shown above (Figure 8) becomes even more significant.
of Arabidopsis MGD1 with the inner chloroplastic envelope
membrane (Block et al., 1983; Miege et al., 1999; Awai et al.,
2001), its exact topology had not been experimentally verified.
We expected that an understanding of the topology of MGD1
would allow us to make a testable prediction regarding its
functional cooperation with TGD1 during lipid metabolism. To-
ward this end, we performed import experiments using in vitro–
translated MGD1 protein and isolated intact chloroplasts. After
import, chloroplasts were reisolated and subjected to protease
treatment with either thermolysin or trypsin in the absence or
presence of detergents. As controls, ARC6 of Arabidopsis (Vitha
et al., 2003) and a truncated version of Tic110 (tp110-110N) from
pea (Pisum sativum; Lu ¨beck et al., 1997) were used in parallel
import and protease protection assays. It has previously been
determined that ARC6 is a protein of the chloroplastic inner
envelope, which is thermolysin resistant but contains a trypsin-
sensitive domain that protrudes into the intermembrane space
(Vitha et al., 2003). By contrast, the bulk of the truncated inner
envelope membrane–localized Tic110-110N faces the stroma,
thus making this truncated membrane protein both thermolysin
and trypsin resistant (Jackson et al., 1998). The results of the
MGD1 import experiments are shown in Figure 9C. The prepro-
tein of MGD1 (Figure 9C, pMGD1) is close in size to the ribulose-
1,5-bisphosphate carboxylase/oxygenase large subunit and
was therefore partially covered up by this most abundant protein
in leaves (Figure 9C, top, lane 1). The MGD1 preprotein was
processed under import conditions to generate mature MGD1
(Figure 9C, mMGD1) and was likewise shown to be resistant to
Figure 8. Incorporation of Lipid Substrates into MGDG by Isolated Wild-
Type and Mutant Chloroplasts.
(A) MGDG biosynthesis from labeled DAG. Additions of UDP-galactose
(UDP-Gal) and phospholipase C (PLC) are indicated.
(B) MGDG biosynthesis from labeled PC. Additions of UDP-Gal and PLC
(C) Time course of MGDG biosynthesis from labeled PA. UDP-Gal and
cytosol were added, and PLC and ER were omitted in this assay.
(D) MGDG biosynthesis from labeled PA. Additions of UDP-Gal, cytosol
fraction, and ER fraction are indicated.
Open symbols or bars represent the wild type and closed symbols or
bars tgd1-1 mutant chloroplasts. In general, three replicates were
averaged, except two in (C), and the standard error is shown.
Altered Phosphatidate Metabolism in tgd13101
trypsin (Figure 9C, top, lane 5). Disruption of the chloroplast by
detergent treatment made the mature MGD1 accessible to
degradation by both thermolysin and trypsin (Figure 9C, top,
lanes 7 to 10). Import assays involving the two control proteins,
ARC6 (Figure 9C, middle), and truncated Tic110-110N (Figure
9C, bottom) behaved in agreement with previously published
results as described by Vitha et al. (2003) and Jackson et al.
(1998), respectively. Briefly, both control proteins were thermo-
lysin resistant. However, while ARC6 was cleaved by trypsin,
truncated Tic110-110N was completely resistant to trypsin
degradation (Figure 9C, bottom, see lane 5). Taken together,
the data presented here support the conclusion that MGD1 is
tightly associated with either the outer or inner chloroplastic
envelope in a peripheral manner with the bulk of the MGD1
protein orientated toward the intermembrane space. Determin-
ing in a more rigorous fashion whether MGD1 peripherally as-
sociates with either the outer or inner envelope was not further
investigated here. However, we have demonstrated that im-
ported mature MGD1 does associate with the total membrane
contains no predicted transmembrane domain(s). Furthermore,
we have confirmed that mature MGD1 can be removed from the
envelope membrane when extracted with either high salt or
sodium carbonate (see Supplemental Figure 3 online). In con-
junction with results obtained by others showing directly that
MGD1 and its ortholog in spinach (Spinacia oleracea) are
associated with the inner envelope membrane (Miege et al.,
1999; Awai et al., 2001), one can conclude that MGD1 is located
on the outside of the inner chloroplastic envelope membrane.
TGD1 Is a Multispanning Integral Envelope
To determine the subcellular localization of TGD1, we stably
expressed a C-terminal fusion of TGD1 with green fluorescent
protein (GFP) under the control of the CaMV 35S promoter and
showed that expression of this fusion protein in Arabidopsis wild
type resulted in a punctate fluorescence pattern at the periphery
of chloroplasts (Figures 10A to 10C). No fluorescence signal was
observed that could be associated with other subcellular mem-
the potentially high expression levels of the TGD1-GFP fusion
protein itself. To address this concern, the abundance of the
TGD1-GFP fusion protein was estimated by probing leaf protein
derived from the transgenic lines with anti-GFP antibodies. We
observed only a weak signal during this analysis (see Supple-
excessively overexpressed in the transgenic lines or in the tissue
examined. It should also be noted that the same TGD1-GFP con-
struct led to a reversion of mutant phenotypes when introduced
Figure 9. MGD1 Activity, Expression, and Localization in the Wild Type
and the tgd1-1 Mutant.
(A) Incorporation of labeled UDP-Gal into the three galactolipids MGDG,
(closed bars). Samples were treated with thermolysin (TL) as indicated.
Three replicates were averaged, and the standard error is shown.
(B) Abundance of MGD1 mRNA in the wild type and the tgd1-1 mutant.
Total RNA was isolated from leaves. The top panel shows an autoradio-
graph of the RNA/DNA hybridization signal; the bottom panel shows one
of the rRNA bands on the ethidium bromide–stained gel that was blotted.
(C) In vitro import of labeled in vitro–translated precursor proteins by pea
chloroplasts. The top panel shows the import of pMGD1, the middle
panel the import of pARC6, and the bottom panel the import of truncated
tpTic110-110N. After import, chloroplasts were subjected to post-
treatment with either thermolysin (TL) or trypsin (TR) in the absence (?)
or presence (þ) of Triton X-100 (TX-100). Intact chloroplasts were
recovered by centrifugation through a 40% Percoll cushion and frac-
tionated into a total membrane (P) and a supernatant (S) fraction. All
fractions were analyzed by SDS-PAGE and fluorography. TP represents
10% of translation product added to an import assay. pMGD1, precursor
of MGD1; mMGD1, mature processed form of MGD1; pARC6, precursor
of ARC6; mARC6, mature form of ARC6; tp110-110N, N-terminal
fragment of Tic110 with transit peptide; 110N, N-terminal fragment of
Tic110, after loss of transit peptide. The bands in lane 5 marked with
asterisks represent cleavage products following trypsin treatment.
3102 The Plant Cell
into the tgd1-1 mutant demonstrating its correct function. Thus,
we assume within reason that the punctate fluorescence pattern
shown in Figure 10 accurately reflects the proper targeting of the
TGD1 protein to the plastid envelope membrane.
Employing an improved in vitro translation system for TGD1,
2003), we examined the in vitro targeting and insertion of
TGD1 into the chloroplast envelope membranes. When in vitro–
translated pTGD1 protein was incubated with isolated pea
chloroplasts under import conditions, we observed that pTGD1
was processed to the mature form, mTGD1 (Figure 10D). After
import and treatment with thermolysin, the mTGD1 form was
membrane fraction. This experiment was done repeatedly, and
in no instance was complete digestion of mTGD1 with thermo-
lysin observed. It should be noted that TGD1 does not contain
a predictable N-terminal chloroplast transit peptide. Thus, TGD1
proper targeting and insertion into the envelope membrane. To
further investigate whether pTGD1 is indeed processed during
import, in vitro–translated pTGD1 was incubated with a crude
chloroplastic stromal preparation that contained the stromal
processing peptidase (SPP), which cleaves and removes transit
peptides from precursor proteins (Tranel and Keegstra, 1996).
Treatment of translated pTGD1 with crude SPP generated a
mTGD1 that was produced from a chloroplastic import assay
(Figure 10E, compare import assay with SPP assay). We con-
clude from this comparison that pTGD1 does contain a potential
nonclassical transit peptide that targets pTGD1 to the envelope
membrane of chloroplasts.
Partial thermolysin sensitivity of mTGD1 observed in the in
vitro import assay system continued to raise questions with
membranes. To complement our in vitro import assays (Figure
10D), an in vivo chloroplast protein import assay was developed
(Figure 10F). For this new assay approach, we generated
C-terminal GFP fusion constructs of TGD1, of DGD1, which lo-
et al., 2001), and of ATS1, the plastidic glycerol-3-phosphate
acyltransferase that is localized to the stroma or on the inside of
theinner chloroplast envelope(Joyard andDouce,1977;Nishida
et al., 1993). The expression of these three fusion proteins
was driven by the CaMV 35S promoter. These constructs were
transiently expressed in tobacco (Nicotiana tabacum) leaves
following Agrobacterium tumefaciens infection. Expression was
enhanced by coexpressing a viral suppressor protein construct
(Voinnet et al., 2003). Once the transgenes were fully expressed,
chloroplasts were isolated and were then treated with thermo-
lysin, trypsin, or buffer (Figure 10F). The GFP tag of these three
fusion proteins was detected by protein gel blot analysis using
GFP antibodies. The result of this experiment is shown in Figure
10F. The TGD1-GFP fusion protein was thermolysin resistant but
trypsin sensitive, suggesting that the GFP portion of the protein
and consequently the C terminus of TGD1 were facing the
intermembrane space. The DGD1-GFP protein was sensitive to
both proteases, as expected for a protein on the outside of the
outer envelope membrane, while the ATS1-GFP fusion was not
cleaved by either protease, as expected for a protein localized
either on the inside of the inner envelope or in the stroma. Based
on these multiple lines of evidence, we conclude that pTGD1
was imported into chloroplasts, processed, and subsequently
targeted to the inner chloroplastic envelope membrane.
Diversion of Lipids at the ER and the Chloroplast
Envelopes in the tgd1 Mutant
At first glance, mutations in TGD1 represented by the chemically
induced alleles tgd1-1, tgd1-2, and tgd1-3 caused a bewildering
Figure 10. Subcellular Localization of TGD1 and Membrane Association.
(A) to (C) Stable transformation of Arabidopsis wild type with a TGD1-
GFP fusion construct. Chlorophyll fluorescence (A), GFP fluorescence
(B), and merged image (C).
(D) In vitro import of pTGD1 into isolated pea chloroplasts. After import of
pTGD1, chloroplasts were subjected to post-treatment with thermolysin
(TL). Intact chloroplasts were recovered by centrifugation through a 40%
Percoll cushion and fractionated into total membrane (P) and supernatant
(S) fractions. All fractions were analyzed by SDS-PAGE and fluorography.
TP represents 10% of translation product added to an import assay. A
fragment appearing after thermolysin treatment is marked by an asterisk.
pTGD1, precursor of TGD1; mTGD1, mature form of TGD1.
(E) pTGD1 was subjected to an SPP assay. An import experiment as
described in (D) is shown for comparison next to the lane showing the
results of an SPP assay of pTGD1.
(F) In vivo import assay of GFP fusion proteins. Top, TGD1-GFP; middle,
DGD1-GFP; bottom, ATS1-GFP. The GFP fusion constructs were tran-
siently expressed in tobacco leaves. Chloroplasts were isolated and
treated either with thermolysin (TL) or trypsin (TR) as indicated. Protein gel
blots are shown on which the GFP portion of the fusion proteins was
Altered Phosphatidate Metabolism in tgd1 3103
of the tgd1-1 mutant revealed that plastidic as well as extra-
plastidic lipid pools were affected. The aberrant production of
lipids not associated with the chloroplast in leaves included
TAGs (Figure 6A), which had a fatty acid composition consistent
with their origin from the eukaryotic pathway of lipid assembly
associated with the ER (Figure 6A). Osmium-dense structures
present in the cytosol of tgd1-1 cells but absent from wild-type
cells (Figure 5) were likely containing these TAGs and presum-
ably represented oil droplets. These TAGs were also found in
nonphotosynthetic tissues, such as roots of the tgd1-1 mutant
(Figure 6A), supporting the notion that they were not associated
with chloroplasts. This was directly shown by isolating chloro-
plasts and demonstrating that they lacked TAGs (Figure 6A).
The substantial accumulation of TAGs in the roots of the tgd1-1
mutant was somewhat surprising because only proplastids are
present in the roots requiring a relatively minor flux through the
eukaryotic pathway of thylakoid lipid biosynthesis during growth
as compared with chloroplasts in the leaves. At this time, we do
in roots of the tgd1-1 mutant. Clarifying this aspect of the mutant
will require the identification and analysis of the enzyme(s)
responsible for the biosynthesis of the root TAGs.
In vegetative tissues of growing, healthy, wild-type plants,
TAGs are usually not present in measurable amounts, but they
accumulate during seed development in the embryo where they
serve as storage compounds. These seed oil TAGs have a dis-
tinct fatty acid composition that includes diagnostic very-long-
chain fatty acids (Voelker and Kinney, 2001). Very-long-chain
fatty acids were absent from TAGs accumulating in tgd1-1 leaf
tissues, distinguishing the phenomenon described here from, for
example, the de novo synthesis of TAGs in green seedlings re-
programmed to synthesize seed TAGs by the expression of the
WRI1 gene that encodes a transcription factor controlling stor-
age oil biosynthesis (Cernac and Benning, 2004). Moreover,
TAGs accumulating in leaves of tgd1-1 were different in their
fatty acid composition from TAGs accumulating in ozone-
fumigated leaves (Sakaki et al., 1990). In this case, TAGs were
similar in fatty acid composition to the predominant thylakoid
derived from the prokaryotic pathway, suggesting a product
precursor relationship between the TAGs and MGDG. Appar-
ently, TAG accumulation in tgd1-1 was not related to a stress-
induced turnover of thylakoid lipids as described for ozone
In addition to TAG accumulation, oligogalactolipids synthe-
sized at the outer chloroplast envelope as a result of the
activation of a processive galactosyltransferase accumulated
in the tgd1-1 mutant (Table 1). These oligogalactolipids showed
a fatty acid composition consistent with their origin from the
prokaryotic pathway associated with the chloroplast (Xu et al.,
2003). While we do not yet understand what controls the ac-
cumulation of TAGs and oligogalactolipids in the tgd1-1 mutant,
it seems likely that eukaryotic lipids are funneled into TAGs at
the ER and prokaryotic lipids into oligogalactolipids at the
chloroplast envelopes. This interpretation would be consistent
with a disruption of the movement of eukaryotic lipids from the
ER to the chloroplast, leading to a backup of eukaryotic lipid
species at the ER and an increased ratio of prokaryotic lipid
species in the chloroplast to make up for the lack of eukaryotic
lipids. One uncertainty is the location of TAG biosynthesis in the
tgd1 mutant. In seeds, TAGs are typically produced by ER-
associated enzymes, and we assume that the same is the case
for TAGs produced in the leaves of the tgd1-1 mutant. The
possibility that the tgd1-1 leaf TAGs are synthesized at the outer
plastid envelope seems highly unlikely, as most of the lipids
produced at the chloroplast envelopes in the tgd1-1 mutant are
of prokaryotic origin, while the TAGs are of eukaryotic origin
based on their fatty acid composition.
PA Is an Intermediate of Eukaryotic MGDG Biosynthesis
The TGD1 protein was predicted to be an integral membrane
component of a multipartite bacterial-type ABC transporter
complex that was proposed to be involved in the eukaryotic
pathway of thylakoid lipid biosynthesis (Xu et al., 2003). The key
question was, what molecule(s) is (are) transported by the
complex such that impairment of the transport complex leads
to the accumulation of TAGs in the cytosol. It seems likely that
TAG itself is not transported, but rather a precursor of TAGs that
backs up at the ER and is funneled into TAGs in the tgd1-1
mutant. The biosynthesis of TAGs draws from intermediates of
phospholipid metabolism, such as PA or PC (Ohlrogge and
Browse, 1995). Indeed, fivefold higher levels of PA were found in
the tgd1-1 mutant, while the relative bulk amounts of other
phospholipids were slightly decreased or remained the same
(Table 1). Because PA is a regulatory molecule in addition to
a central metabolite, one must assume that in the tgd1-1 mutant,
excess PA at the ER is converted primarily into TAG, which can
be harmlessly sequestered into oil droplets as shown in Figure 5.
Interestingly, we did not observe an accumulation of PC in the
mutant (Table 1). Likewise, an obvious increase in the content of
DAG, which is the intermediate in the conversion of PA to MGDG
and presumably TAG in the mutant, was not apparent in most
experiments. The fact that PC did not accumulate suggests that
PA and not PC might be the eukaryotic lipid transported from the
ER to the envelopes.
Because of the consistent increase in PA content in the tgd1-1
mutant, the question arose whether PA could be the substrate of
the TGD1 transport complex. Comparative labeling experiments
with isolated wild-type and tgd1-1 mutant chloroplasts showed
an impairment of conversion of label fromPC and PA into MGDG
in mutant chloroplasts (Figure 8). The rates for PC and PA
labeling were similar and were approximately half of those
observed for DAG, which is the direct substrate of MGD1, the
main enzyme responsible for MGDG biosynthesis in Arabidopsis
(Jarvis et al., 2000). A caveat in the interpretation of these
experiments was the increased MDG1 activity in the tgd1-1
mutant, visible when labeled DAG (Figure 8A) orUDP-Gal (Figure
9A) was incubated with chloroplasts. However, because PA
incorporation was decreased in mutant chloroplasts in spite of
limiting in these experiments. The reason for the activation of
MGD1 in the mutant is not known, but activation might be
brought about by a changed lipid environment at the inner
3104 The Plant Cell
Adding labeled PC and phospholipase C to the reactions,
which directly generates DAG from PC, did stimulate the in-
corporation of label into MGDG and nearly abolished the differ-
ences between the mutant and the wild type (Figure 8B). This
result suggested that PA is the intermediate in the conversion of
PC to DAG, in which TGD1 plays a role. Presumably, when only
labeled PC is added to chloroplast preparations, anendogenous
phospholipase converts it to PA, which is further metabolized to
MGDG, but at a slower rate in the mutant chloroplasts. It should
be noted that in similar experiments with isolated pea chloro-
plasts, a stimulation of incorporation of labeled PC by cytosolic
fractions was observed and was attributed to cytosolic phos-
pholipases producing PA (Andersson et al., 2004). The stimula-
tion of PA incorporation following addition of cytosol fractions in
are required under the employed conditions for a possible trans-
fer of PA offered in the form of liposomes to the chloroplasts.
Stimulation was not observed for the mutant presumably be-
cause the activity of the TGD1 mutant protein was limiting for the
incorporation of PA into MGDG or because these factors could
not interact with the TGD1 mutant protein. In any case, a direct
conclusion from the results of this comparative analysis was that
PA mediated by TGD1 is a precursor of MGDG biosynthesis.
Moreover, PA as it is synthesized at the ER could possibly be the
direct substrate provided by the ER for eukaryotic MGDG bio-
synthesis. This latter suggestion and the possible involvement of
TGD1 are supported by the accumulation of PA but not PC in the
tgd1-1 mutant. However, our current data do not rule out that PC
is transferred from the ER to the outer chloroplast envelope,
where it could be converted to PA by a not yet identified enzyme,
making it available to the TGD1 complex. Most likely, PC would
have to be transferred from the ER to the chloroplast even if PA
were the preferred ER lipid for the biosynthesis of eukaryotic
MGDG because there is no known PC biosynthetic activity
associated with the chloroplast envelopes (Douce and Joyard,
What Is the Biochemical Function of TGD1 at the Inner
To understand the role that TGD1 might play in the conversion of
PA to DAG, it was essential to determine the subcellular location
of this protein within chloroplasts. An analysis of the amino acid
sequence of TGD1 revealed that this protein does not contain
a predictable transit peptide. Previous in vitro import and pro-
tease protection assays showed that TGD1 is partially thermo-
lysin sensitive, leadingto the tentative conclusion thatitmight be
2003). However, upon reinvestigating the subcellular localization
of TGD1 using several alternative approaches, we obtained
multiple lines of evidence (Figure 10) suggesting that this protein
most likely is located in the inner chloroplastic envelope mem-
brane. For instance, to complement our standard in vitro chlo-
roplastic import assays, we developed an in vivo import assay
using the transient expression of proteins in tobacco leaves
followed by the isolation and protease treatment of chloroplasts.
The results presented in Figure 10F show that after import and
but trypsin sensitive. This result disagrees with the results
derived from the in vitro import assays (Figure 10D). One
interpretation of this discrepancy may be that in our newly
developed in vivo import assay system, conditions are favorable
for the proper and complete insertion of the TGD1-GFP fusion
protein into the envelope membrane. In contrast with our in vitro
import assay system, chloroplasts might have difficulty import-
ing and subsequently inserting a multispanning membrane
protein into the chloroplastic envelope membrane. Indeed,
discrepancies between in vitro and in vivo import systems have
been reported previously (Silva-Filho et al., 1997). Likewise,
attempts to employ in vitro import assay systems to investigate
membrane proteins with multiple transmembrane domains,
such as chloroplastic metabolite translocators, have proven to
be very problematic and challenging (Schunemann et al., 1993;
Fischer et al., 1994; Weber et al., 1995; Flu ¨gge, 1998). Hence, to
overcome these technical difficulties, we employed several
strategies to examine the import and localization of TGD1 within
chloroplasts. Additionally, the in vivo import assays also pro-
vided added information on the possible topology of TGD1,
suggesting that the C-terminal end of TGD1 may be orientated
toward the intermembrane space (Figure 10F). The currently
accumulated evidence suggests that TGD1 is in fact being
inserted into the inner chloroplastic envelope membrane. How-
ever, the partial thermolysin sensitivity of TGD1 in import assays
leaves the question open whether this protein might actually
cross the inner and outer envelopes, a difficult to prove hypoth-
esis at this time.
In light of our revised localization of TGD1 and assuming that
TGD1 might facilitate PA transfer through the inner envelope, the
question arises why a PA transporter at the inner chloroplastic
envelope would be necessary for the biosynthesis of eukaryotic
MGDG given that we and others have shown that the major
MGDG synthase, MGD1, is tethered to the outside of the inner
envelope in a peripheral manner protruding into the intermem-
brane space (Figure 9C; see also Miege et al., 1999; Awai et al.,
2001). The answer can be found in the apparent localization of
PAP on the inside of the inner envelope. Evidence for the
exclusive and tight association of chloroplast PAP activity with
the inner envelope has been published (Block et al., 1983;
Andrews and Mudd, 1985). While these experiments could not
distinguish whether PAP was on the inside or outside of the inner
is affected by Mg2þsimilar to other stroma proteins would be in
agreement with a location on the inside of the inner envelope
chloroplast envelopes lack phospholipase C activity (Roughan
and Slack, 1982; Browse and Somerville, 1991; Marechal et al.,
1997), which could directly produce DAG from PC. Since PA by
itself cannot readily traverse the membrane, a PA transporter at
the inner chloroplast envelope would be a prerequisite for the
conversion of eukaryotic PA into DAG by the PAP on the inside
of the inner envelope. While we have no proof at this time that
TGD1 and associated factors directly transfer PA through the
inner chloroplast envelope, the underlying working hypothesis
implies that the eukaryotic and prokaryotic pathways share the
plastidic PAP. The activity of this enzyme is much lower in plants
that exclusively use the eukaryotic pathway for galactolipid
Altered Phosphatidate Metabolism in tgd13105
both pathways (Heinz and Roughan, 1983). A critical test of this
hypothesis will have to await the identification of the gene for
plastidic PAP. Furthermore, as we expect that additional
components of the transporter exist in Arabidopsis based on
additional tgd1-like nonallelic mutants (Xu et al., 2003), a re-
constitution and direct demonstration of PA transport mediated
by the TGD1 complex is not yet possible, and will have to await
the isolation of all components.
Bringing together the eukaryotic and prokaryotic pathways at
the level of PAP is consistent with the fact that only a single
MGDG synthase, MGD1, is responsible for the biosynthesis of
the bulk of eukaryotic and prokaryotic lipids (Jarvis et al., 2000).
How DAG produced on the inside of the inner envelope moves to
not known. Furthermore, the processes by which MGDG pro-
duced by MGD1 moves to DGD1 on the outside of the outer
TGD1 Function Appears to Be Essential
allele, tgd1-1, frequently led to an abortion of developing seeds
at the heart stage, when greening of the chloroplast occurs in
enhanced by reducing the level of tgd1-1 mRNA in an RNAi
experiment (Figure 3). Fatty acids in MGDG with 16 carbons, in
respective MGDG species (Heinz and Roughan, 1983). In Arabi-
dopsis leaves, ;50% of the MGDG molecules are derived from
the prokaryotic pathway, and a substantial fraction of fatty acids
in MGDG is of the 16-carbon type (Browse et al., 1986). In-
terestingly, in developing seeds, the fatty acid profile of MGDG,
which is a marker lipid for the plastid, showed much less 16-
carbon fatty acids than MGDG in leaves (Figure 4D), suggesting
that most of MGDG in embryo chloroplasts is derived from the
eukaryotic pathway. The metabolism in embryos is specialized
toward the biosynthesis of TAGs, which are exclusively derived
from the eukaryotic pathway (Ohlrogge and Browse, 1995).
Apparently, this prevalence of the eukaryotic pathway in em-
bryos extends to the thylakoid lipids in the chloroplasts. If TGD1
plays an essential role in the biosynthesis of eukaryotic plastid
lipids, embryos affected in TGD1 function cannot produce
functional plastids and abort. Alternatively, the possible accu-
mulation of PA, a potential signaling molecule, in the tgd1-1
mutants simply might not be tolerated. As a consequence, seed
abortion was observed, although with varying penetrance in the
different tgd1-1 mutant alleles and stronger in the tgd1-1/TGD1-
RNAi lines. Varying penetrance of seed abortion is frequently
encountered in mutants affected in primary lipid metabolism
(Bonaventure et al., 2004). When the amount of TAGs in mature
homozygous tgd1-1 seeds that did develop was determined, no
change in oil content per seed was observed. This result was in
agreement with a proposed function of TGD1 as a PA trans-
locator at the inner chloroplast envelope, which would presum-
ably play no role in seed TAG biosynthesis at the ER. While the
is also important for the growing plant because growth of the
tgd1-1 mutant was slightly reduced and more so for the tgd1-1/
plays an essential role throughout the life cycle of the plant
consistent with its hypothesized involvement in the transport of
a central metabolite of primary lipid metabolism and a regulatory
molecule, such as PA, into the chloroplast.
Plant Materials and Growth Conditions
The Arabidopsis thaliana tgd1-1 mutant allele, which was originally
isolated in the dgd1 background (Xu et al., 2003), was crossed into the
wild-type background (ecotype Columbia-2) and at least three times
backcrossed. Unless otherwise indicated, all experiments were con-
ducted with this line. The act1(ats1) mutant has been previously de-
of the TGD1 locus was done as previously described (Xu et al., 2003).
Surface-sterilized seeds were germinated on 0.8% (w/v) agar-solidified
MS medium (Murashige and Skoog, 1962) supplemented with 1% (w/v)
sucrose. Typically, 10-d-old seedlings of Arabidopsis wild type and
mutants were transferred to soil drenched with half-strength Arabidopsis
nutrient solution (Estelle and Somerville, 1987) and grown under a pho-
tosynthetic photon flux density of 70 to 80 mmol m?2s?1at 22/188C (day/
night) with a 14-h-light/10-h-dark period. For quantitative growth experi-
shelf of a CU-36L5 growth chamber (Percival Scientific). At each time
point, aerial parts of 15 wild-type and mutant plants on matching shelf
positions were weighed. For root growth assays, 10 seedlings for each
genotype were grown on vertically positioned agar plates, and 10
seedlings were analyzed per time point. Standard errors were calculated.
Generation of RNAi Lines
The full-length coding region of TGD1 was amplified by PCR using the
primers 59-AATACTAGTGGCGCGCCATGATGCAGACTTGTT-39 (SpeI
and AscI; sense primer) and 59-CCAGGATCCATTTAAATTCAAACA-
CAGTTCTT-39 (BamHI and SwaI; antisense primer) and the original
plasmid as template (Xu et al., 2003). The resulting DNA fragment was
cloned into the silencing vector pGSA1285 (ABRC, Ohio State University,
Columbus, OH; CD3-454) in sense and antisense orientations separated
by a glucuronidase intron and was expressed under the control of the
CaMV 35S promoter. Plant transformation was achieved by the floral dip
method (Clough and Bent, 1998). Resistant seedlings were selected on
MS medium containing 40 mg/mL of kanamycin.
Leaves and siliques were imaged using a Leica MZ 12.5 dissecting
microscope (Leica Microsystems) equipped with a Spot Insight color
camera (Diagnostic Instruments). Siliques from homozygous tgd1-1
plants were dissected with hypodermic needles. Seeds were cleared in
chloral hydrate solution (chloral hydrate:water:glycerol, 8:2:1 w/v/v) for
1 h at room temperature and were observed with a Leica DMLB micro-
scope equipped with Nomarski optics (Leica Microsystems).
For electron microscopy, leaf tissues from 3-week-old wild-type and
tgd1-1 mutant plants grown on agar-solidified medium were fixed for 2 h
at room temperature in 2.5% glutaraldehyde and 0.1 M sodium phos-
phate, pH 7.2, followed by a secondary fixation in 1% (w/v) osmium
tetroxide in the same buffer. After this double fixation, samples were
3106 The Plant Cell
dehydrated in a graded series of acetone, embedded in EPON812 resin
(Electron Microscopy Sciences), and sectioned. The thin sections (;70
to 90 nm) were stained with uranyl acetate and lead citrate prior to
examination in a JEOL 100CX electron microscope (JEOL).
For the GFP fusion protein localization study, leaf samples were
mounted in water on slides and were directly examined using a Zeiss
LSM5 confocal microscope. Excitation light was provided by an argon
laser at 488 nm. GFP fluorescence was observed with a band-pass filter
of 505 to 530 nm and chlorophyll fluorescence with a 650-nm long-pass
filter. Enhanced-quality images were acquired with the LSM5 imaging
system software, and postacquisition image processing was performed
with the LSM5 image browser and Adobe Photoshop software. This work
was performed at the Center for Advanced Microscopy (Michigan State
Lipid and Fatty Acid Analyses
Lipids were extracted as previously described (Do ¨rmann et al., 1995).
Polar lipid extracts were analyzed on activated ammonium sulfate–
impregnated silica gel TLC plates (Si250 with preadsorbent layer;
Mallinckrodt Baker) using a solvent system of acetone:toluene:water
(91:30:7, v/v). Two-dimensional TLC was used to separate PA as pre-
viously described (Benning et al., 1995). Neutral lipids were separated on
the same silica plates, but untreated with ammonium sulfate, using
a solvent system consisting of petroleum ether:ethyl ether:acetic acid
(80:20:1, by volume). Lipids were visualized with iodine vapor and
identified by cochromatography with lipid extracts of known composition
or commercially purchased standards. For quantitative analysis, individ-
ual lipids were isolated from TLC plates and used to prepare fatty acid
methyl esters. The methyl esters were quantified by gas–liquid chroma-
tography using myristic acid as internal standard (Rossak et al., 1997).
Positional analysis of fatty acids in PA was done as previously described
(Ha ¨rtel et al., 2000).
For in vivo labeling experiments, detached leaves from 3-week-old
Arabidopsis plants were incubated with 3.7 MBq carrier-free [32P]ortho-
phosphate (Amersham) in 20 mL of MS medium. Lipids were extracted
above. Radiolabeled lipids were visualized by autoradiography.
Isolation of ER Membrane and Cytosol Fraction
ER-enriched membranes were isolated from 3-week-old seedlings ac-
cording to Bessoule et al. (1995). The resulting ER membranes were
suspended in incubation buffer (see below). Cytosol fraction was isolated
according to Andersson et al. (2004) with some modifications. Seedlings
were homogenized in 50 mL of 50 mM HEPES-KOH, pH 7.0, 10 mM KCl,
2.5 mM MgCl2, and 5 mM ascorbate and supplemented with 1 mL of
protease inhibitor cocktail for plant extracts (P 9599; Sigma-Aldrich). The
homogenate was then filtered through two layers of Miracloth (Calbio-
chem) and centrifuged at 12,000g for 10 min, and the supernatant was
centrifuged further at 100,000g for 60 min. The supernatant was con-
centrated 10 times with a centricon 10 kD cutoff filter (Millipore). The
concentrated cytosol fraction was frozen in liquid nitrogen and stored at
?808C until further use.
Chloroplast Lipid Import Assays
Intact chloroplasts were isolated from 3-week-old Arabidopsis plants by
discontinuous gradient (5 mL 80% and 10 mL 40%) as previously
described (Xu et al., 2002). Pigments were quantified according to
Radioactive PC for chloroplast labeling assays was prepared after
incubating 16-d-old Arabidopsis seedlings overnight in 20 mM MES-
KOH, pH 6.0, with 50 mmol of [1-14C]-acetate (2.22 GBq/mmol; American
Radiolabeled Chemicals) according to Kelly et al. (2003). Lipids were
extracted and separated by TLC. Radioactive PC was eluted from silica
gel using chloroform:methanol:formic acid (10:10:1, v/v) and redissolved
in chloroform:methanol (3:1, v/v). The [14C]-labeled PA and DAG sub-
strates were prepared by digesting radiolabeled PC with phospholipase
D (Type I, cabbage [Brassica capitata]) and phospholipase C (Type XI,
Bacillus cereus), respectively. All enzymes were from Sigma-Aldrich.
Specific radioactivity of the resulting [14C]-PC, -PA, and -DAG were
determined by gas chromatography and scintillation counting and were
;42, 16, and 12 MBq/mmol, respectively.
The chloroplast labeling experiments were done according to Ohnishi
of radiolabeled [14C]-lipids and 10 nmol of unlabeled lipids dissolved
in chloroform:methanol (2:1, v/v), were added to a glass vial, and the
solvent was evaporated under a steam of nitrogen. Lipids were solubi-
lized by sonication in 100 mL of incubation buffer (0.33 M sorbitol, 50 mM
HEPES-KOH, pH 7.0, 10 mM KCl, 1 mM ATP, and 2.5 mM MgCl2). In
some experiments, as indicated in the figure legends, 0.3 units of phos-
pholipase C or unlabeled UDP-Gal at a final concentration of 2 mM were
protein each. The reaction was started by addition of the chloroplast
preparation (50 mg chlorophyll) and was stopped after 30 min if not
Lipids were separated by TLC as described above. After staining with
iodine vapor, lipids were scraped off the plate and counted by liquid
MGD1 Activity Assays
Intact Arabidopsis chloroplasts were isolated as above. In some experi-
ments, the purified chloroplasts (1 mg/mL chlorophyll) were incubated
with 0.5 mg/mL thermolysin (Sigma-Aldrich) in 0.33 M sorbitol, 50 mM
HEPES, pH 7.3, and 1.0 mM CaCl2in the dark for 30 min at 48C. After
treatment, intact chloroplasts were repurified by centrifugation through
in 0.33 M sorbitol, 50 mM HEPES, pH 7.6, 5 mM EDTA, 1 mM MnCl2, and
1 mM MgCl2. Total galactolipid synthesis in intact chloroplasts (50 mg
chlorophyll) was assayed by incubation with 18.5 KBq of UDP-[U-14C]-
galactose (10.28 GBq/mmol; American Radiolabeled Chemicals) in
150 mL of medium as described above for resuspension. Lipids were
extracted and separated by TLC, and the radiolabel was quantified as
PAP Activity Assay
Mixed envelope membranes from purified wild-type and tgd1-1 chloro-
plasts were prepared by the method of Froehlich et al. (2003). Activity of
PAP was assayed by following PA conversion into DAG by isolated
envelope membranes according to Malherbe et al. (1992). Radioacitive
PA was synthesized within the envelope membranes by acylation of sn-
Lipids were extracted from aliquots of the reaction mixture taken at
different time intervals, and the neutral lipids were analyzed by TLC as
described above. Radioactivity in DAG was quantified by isolating the
respective silica area from the plate followed by scintillation counting.
Quantification of RNA
Total RNA was isolated using Trizol reagent (Invitrogen) according to the
manufacturer’s instructions. For RNA gel blots, RNA was separated by
agarose gel electrophoresis and blotted onto Hybond Nþmembranes
(Amersham) using standard procedures and standard high-stringency
Altered Phosphatidate Metabolism in tgd1 3107
For RT-PCR quantification of TGD1 RNA, total RNA was extracted from
3-week-old plants using the RNeasy plant mini kit (Qiagen) following the
First-strand cDNA was synthesized using the Ominiscript reverse tran-
scription kit (Qiagen). PCR thermocycling conditions were 948C for 3 min
followed by 29 cycles of 948C for 30 s, 558C for 30 s, and 728C for 1 min,
with a final polymerization step at 728C for 10 min. Primers used to amplify
a 726-bp fragment of the TGD1 gene were 59-CAAGGTACCATGATGCA-
GACTTGTTGTAT-39 (forward) and 59-TCAGAAACACATGAGTGTCAG-39
(reverse). A 317-bp fragment within the ubiquitin UBQ10 gene (GenBank
At5g05320) was amplified separately as a control. The UBQ10 forward
primer was 59-TCAATTCTCTCTACCGTGATCAAGATGCA-39, and the re-
verse primer was 59-GTGTCAGAACTCTCCACCTCAAGAGTA-39.
In Vitro Chloroplast Import Assays and Stromal
Plasmids expressing cDNAs encoding ARC6 (Vitha et al., 2003), tp110-
110N (Lu ¨beck et al., 1997), and MGD1 (Awai et al., 2001) have been
described previously. These genes were transcribed/translated, and
proteins were subsequently labeled with [35S]-Met using the TNT-coupled
reticulocyte lysate system according to the manufacturer’s recommen-
dations (Promega). The TGD1 cDNA was PCR amplified from the original
expression from the SP6 promoter (Promega). This TGD1 plasmid was
linearized prior to translation with the SP6 RNA Polymerase TNT-coupled
reticulocyte lysate system. Pea plants (Pisum sativum var Little Marvel;
Olds Seed Co.) were grown under natural light in the greenhouse at 18 to
208C. Chloroplasts were isolated from 8- to 12-d-old plants as described
previously (Bruce et al., 1994). Binding or import reactions were per-
formed according to Tranel et al. (1995). Post-treatments of import
reactions with either thermolysin or trypsin were performed as described
previously (Jackson et al., 1998). SPP assays were performed according
to Tranel and Keegstra (1996). All fractions were analyzed by SDS-PAGE
(Laemmli, 1970) and fluorography (Tranel et al., 1995).
Gene-GFP Fusion Constructs
For the generation of a GFP fusion construct, the entire coding region of
(www.cambia.org) using primers 59-AGCCATGGTAATGACTCTCAC-
GTTTTC-39 and 59-CGACTAGTATTCCAAGGTTGTGACAAAG-39. The
N-terminal part of DGD1 up to Pro337was amplified from pBinAR-Hyg-
DGD1 (Do ¨rmann et al., 1999) using primers 59-CACGGTACCATGG-
TAAAGGAAACT-39 and 59-CTCGGATCCAGGCTTCACAAAATC-39 and
was directly cloned into pCambia1300MCS-GFP (a derivative of pCAM-
BIA1302 with a more versatile multiple cloning site in the expression
cassette). This N-terminal portion of DGD1 contains the correct targeting
information and is inserted into the outer envelope membrane as pre-
viously shown. The full-length coding sequence of TGD1, including the
transit peptide, was amplified by PCR using the primers 59-CAGGAG-
ATCTAATGATGCAGACTTGTTG-39 and 59-CCGGACTAGTAACACAGT-
TCTTCAAAGA-39 and pBINAR-Hyg-TGD1 (Xu et al., 2003) as template.
This fragment was inserted into the binary vector pCAMBIA1302 using
the BglI and SpeI restriction sites. Stable transformation of Arabidopsis
was achieved using the floral dip method (Clough and Bent, 1998).
Transgenic plants were selected in the presence of Hygromycin B (25 mg
mL?1) on MS medium lacking sucrose.
In Vivo Chloroplast Import Assays
For transient expressionof GFP constructs in Nicotiana benthamiana,the
Agrobacterium tumefaciens strain C58C1 carrying the respective con-
struct (see above) was injected into the abaxial air space of 3- to 4-week-
old plants according to Voinnet et al. (2003). A second construct
expressing the p19 protein of tomato bushy stunt virus was used to
suppress gene silencing and therefore enhance the level of transient
expression of the target gene construct (Voinnet et al., 2003). Agro-
bacterium strains carrying GFP constructs or the p19 silencing plasmid
were grown to an OD600of 0.5 and 1.0, respectively, prior to mixing of
equal parts and injection. At 5 d after infiltration, leaves were harvested,
and intact chloroplasts were isolated by discontinuous gradient as
described above. Protease protection assays were done according to
McAndrew et al. (2001). Briefly, isolated chloroplasts were treated with
either trypsin or thermolysin at final concentrations of 500 or 800 mg/mL
for 30mininice-cold reactionbuffer(50mMHEPES-KOH, pH7.9,0.33M
sorbitol, and 1 mM CaCl2in a total volume of 250 mL). Intact chloroplasts
were reisolated by centrifugation through 40% Percoll (v/v), washed, and
solubilized directly in sample buffer prior to analysis by SDS-PAGE.
Protein Extraction and Immunoblotting
For GFP fusion protein extraction, 0.1 g of frozen leaf tissue was extracted
with 0.2 mL buffer containing 4 M urea and 100 mM DTT. Two hundred
microliters of loading buffer (Laemmli, 1970) were added and the samples
wereboiled for 5 min. After centrifugation for 5 min at 10,000g, the proteins
immunodetection, a rabbit anti-GFP antibody (Molecular Probes) and anti-
rabbit alkalinephosphatase-coupled antibody (Jackson Immuno Research
Laboratories)were usedat1:1000and 1:10,000dilutions,respectively.The
Arabidopsis Genome Initiative locus identifiers (www.arabiodpsis.org) for
the Arabidopsis genes mentioned in the text are as follows: ACT1(ATS1),
At1g32200; ARC6, At5g42480; DGD1, At3g11670; MGD1, At4g31780;
The following materials are available in the online version of this article.
Supplemental Figure 1. Digestion of Leaf TAG and Soy Oil TAG with
Supplemental Figure 2. Phosphatidate Phosphatase Activity in Iso-
lated Chloroplasts of the tgd1-1 Mutant and the Wild Type.
Supplemental Figure 3. Sodium Chloride and Sodium Carbonate
Extraction of Isolated Pea Chloroplast Total Membranes following
Import of MGD1.
Supplemental Figure 4. GFP Immunoblot of Leaf Extracts of the
Wild Type or the Strongest TGD1-GFP Expressing Transgenic Line
We thank Ken Keegstra, who allowed portions of this research to be
performed in his laboratory at the Michigan State University Department of
Energy, Plant Research Laboratory (East Lansing, MI), David Baulcombe
from the Sainsbury Laboratory (John Innes Centre, Norwich, UK) for
providing the p19 plasmid, and Hiroyuki Ohta from the Tokyo Institute of
Technology (Yokohama, Japan) for providing the MGD1 expression
plasmid. We are grateful to John Ohlrogge (Michigan State University)
and John Browse (Washington State University, Pullman, WA) for dis-
cussions and their constructive criticism. This work was funded in part by
grants to C.B. from the U.S. Department of Energy (DE-FG02-98ER20305)
and from the U.S. National Science Foundation (MCB-0453858). K.A. was
supported by Japan Society for the Promotion of Sciences Postdoctoral
3108The Plant Cell
Fellowships for Research Abroad from the Ministry of Education, Sports,
Science, and Culture of Japan. J.E.F. was supported in part by grants
from the U.S. National Science Foundation (MCB-0316262) and by the
U.S. Department of Energy, Division of Energy Biosciences.
Received June 28, 2005; revised August 24, 2005; accepted September
9, 2005; published September 30, 2005.
Andersson, M.X., Kjellberg, J.M., and Sandelius, A.S. (2004). The
involvement of cytosolic lipases in converting phosphatidyl choline to
substrate for galactolipid synthesis in the chloroplast envelope.
Biochim. Biophys. Acta 1684, 46–53.
Andrews, J., and Mudd, J.B. (1985). Phosphatidylglycerol synthesis
in pea chloroplasts: Pathway and localization. Plant Physiol. 79,
Awai, K., Marechal, E., Block, M.A., Brun, D., Masuda, T., Shimada,
H., Takamiya, K., Ohta, H., and Joyard, J. (2001). Two types of
MGDG synthase genes, found widely in both 16:3 and 18:3 plants,
differentially mediate galactolipid syntheses in photosynthetic and
nonphotosynthetic tissues in Arabidopsis thaliana. Proc. Natl. Acad.
Sci. USA 98, 10960–10965.
Benning, C., Huang, Z.H., and Gage, D.A. (1995). Accumulation of a
novel glycolipid and a betaine lipid in cells of Rhodobacter sphaer-
oides grown under phosphate limitation. Arch. Biochem. Biophys.
Benning, C., and Ohta, H. (2005). Three enzyme systems for galacto-
glycerolipid biosynthesis are coordinately regulated in plants. J. Biol.
Chem. 280, 2397–2400.
Bessoule, J.J., Testet, E., and Cassagne, C. (1995). Synthesis of
phosphatidylcholine in the chloroplast envelope after import of
lysophosphatidylcholine from endoplasmic reticulum membranes.
Eur. J. Biochem. 228, 490–497.
Block, M.A., Dorne, A.J., Joyard, J., and Douce, R. (1983). Prepara-
tion and characterization of membrane fractions enriched in outer and
inner envelope membranes from spinach chloroplasts. II. Biochemical
characterization. J. Biol. Chem. 258, 13281–13286.
Bonaventure, G., Bao, X., Ohlrogge, J., and Pollard, M. (2004).
Metabolic responses to the reduction in palmitate caused by disrup-
tion of the FATB gene in Arabidopsis. Plant Physiol. 135, 1269–1279.
Browse, J., and Somerville, C. (1991). Glycerolipid biosynthesis:
Biochemistry and regulation. Annu. Rev. Plant Physiol. Plant Mol.
Biol. 42, 467–506.
Browse,J.,Warwick,N.,Somerville,C.R.,andSlack,C.R. (1986). Fluxes
through the prokaryotic and eukaryotic pathways of lipid synthesis in
the ‘‘16:3’’ plant Arabidopsis thaliana. Biochem. J. 235, 25–31.
Bruce, B.D., Perry, S., Froehlich, J., and Keegstra, K. (1994). In vitro
import of protein into chloroplasts. In Plant Molecular Biology Manual,
S.B. Gelvin and R.A. Schilperoort, eds (Boston: Kluwer Academic
Publishers), pp. 1–15.
Cernac, A., and Benning, C. (2004). WRINKLED1 encodes an AP2/
EREB domain protein involved in the control of storage compound
biosynthesis in Arabidopsis. Plant J. 40, 575–585.
Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified method
for Agrobacterium-mediated transformation of Arabidopsis thaliana.
Plant J. 16, 735–743.
Do ¨rmann, P., Balbo, I., and Benning, C. (1999). Arabidopsis galacto-
lipid biosynthesis and lipid trafficking mediated by DGD1. Science
Do ¨rmann, P., and Benning, C. (2002). Galactolipids rule in seed plants.
Trends Plant Sci. 7, 112–118.
Do ¨rmann, P., Hoffmann-Benning, S., Balbo, I., and Benning, C. (1995).
Isolation and characterization of an Arabidopsis mutant deficient in
the thylakoid lipid digalactosyl diacylglycerol. Plant Cell 7, 1801–1810.
Douce, R., and Joyard, J. (1996). Biosynthesis of thylakoid membrane
lipids. In Oxygenic Photosynthesis: The Light Reactions, D.R. Ort and
C.F. Yocum, eds (Dordrecht, The Netherlands: Kluwer Academic
Publishers), pp. 69–101.
Estelle, M.A., and Somerville, C. (1987). Auxin-resistant mutants of
Arabidopsis thaliana with an altered morphology. Mol. Gen. Genet.
Fischer, K., Weber, A., Arbinger, B., Brink, S., Eckerskorn, C., and
Flu ¨gge, U.I. (1994). The 24 kDa outer envelope membrane protein
from spinach chloroplasts: Molecular cloning, in vivo expression and
import pathway of a protein with unusual properties. Plant Mol. Biol.
Flu ¨gge, U.I. (1998). Metabolite transporters in plastids. Curr. Opin. Plant
Biol. 1, 201–206.
Froehlich, J.E., Benning, C., and Do ¨rmann, P. (2001). The digalacto-
syldiacylglycerol (DGDG) synthase DGD1 is inserted into the outer
envelope membrane of chloroplasts in a manner independent of the
general import pathway and does not depend on direct interaction
with monogalactosyldiacylglycerol synthase for DGDG biosynthesis.
J. Biol. Chem. 276, 31806–31812.
Froehlich, J.E., Wilkerson, C.G., Ray, W.K., McAndrew, R.S.,
Osteryoung, K.W., Gage, D.A., and Phinney, B.S. (2003). Proteomic
study of the Arabidopsis thaliana chloroplastic envelope membrane
utilizing alternatives to traditional two-dimensional electrophoresis.
J. Proteome Res. 2, 413–425.
Ha ¨rtel, H., Do ¨rmann, P., and Benning, C. (2000). DGD1-independent
biosynthesis of extraplastidic galactolipids following phosphate dep-
rivation in Arabidopsis. Proc. Natl. Acad. Sci. USA 97, 10649–10654.
Heinz, E. (1977). Enzymatic reactions in galactolipid biosynthesis. In
Lipids and Lipid Polymers in Higher Plants, M. Tevini and H.K.
Lichtenthaler, eds (Berlin: Springer-Verlag), pp. 102–120.
Heinz, E., and Roughan, G. (1983). Similarities and differences in lipid
metabolism of chloroplasts isolated from 18:3 and 16:3 plants. Plant
Physiol. 72, 273–279.
Jackson, D.T., Froehlich, J.E., and Keegstra, K. (1998). The hydro-
philic domain of Tic110, an inner envelope membrane component of
the chloroplastic protein translocation apparatus, faces the stromal
compartment. J. Biol. Chem. 273, 16583–16588.
Jarvis, P., Do ¨rmann, P., Peto, C.A., Lutes, J., Benning, C., and
Chory, J. (2000). Galactolipid deficiency and abnormal chloroplast
development in the Arabidopsis MGD synthase 1 mutant. Proc. Natl.
Acad. Sci. USA 97, 8175–8179.
Joyard, J., and Douce, R. (1977). Site of synthesis of phosphatidic acid
and idacylglcyerol in spinach chloroplasts. Biochim. Biophys. Acta
Kelly, A.A., Froehlich, J.E., and Do ¨rmann, P. (2003). Disruption of
the two digalactosyldiacylglycerol synthase genes DGD1 and DGD2
in Arabidopsis reveals the existence of an additional enzyme of
galactolipid synthesis. Plant Cell 15, 2694–2706.
Kunst, L., Browse, J., and Somerville, C. (1988). Altered regulation of
lipid biosynthesis in a mutant Arabidopsis deficient in chloroplast
glycerol-3-phosphate acyltransferase activity. Proc. Natl. Acad. Sci.
USA 85, 4143–4147.
Laemmli, U.K. (1970). Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227, 680–685.
Lee, S., Park, J., and Lee, Y. (2003). Phosphatidic acid induces actin
polymerization by activating protein kinases in soybean cells. Mol.
Cells 15, 313–319.
Altered Phosphatidate Metabolism in tgd13109
Li, W., Li, M., Zhang, W., Welti, R., and Wang, X. (2004). The plasma
membrane-bound phospholipase Ddelta enhances freezing tolerance
in Arabidopsis thaliana. Nat. Biotechnol. 22, 427–433.
Lichtenthaler, H.K. (1987). Chlorophylls and carotenoids: Pigments of
photosynthetic membranes. Methods Enzymol. 148, 350–382.
Loewen, C.J., Gaspar, M.L., Jesch, S.A., Delon, C., Ktistakis, N.T.,
Henry, S.A., and Levine, T.P. (2004). Phospholipid metabolism
regulated by a transcription factor sensing phosphatidic acid. Science
Lu ¨beck, J., Heins, L., and Soll, J. (1997). A nuclear-coded chloroplastic
inner envelope membrane protein uses a soluble sorting intermediate
upon import into the organelle. J. Cell Biol. 137, 1279–1286.
Malherbe, A., Block, M.A., Joyard, J., and Douce, R. (1992). Feed-
back inhibition of phosphatidate phosphatase from spinach chloro-
plast envelope membranes by diacylglycerol. J. Biol. Chem. 267,
Mansfield, S.G., and Briarty, L.G. (1991). Early embryogenesis in Arabi-
dopsis thaliana. II. The developing embryo. Can. J. Bot. 69, 461–476.
Marechal, E., Block, M.A., Dorne, A.-J., and Joyard, J. (1997). Lipid
synthesis and metabolism in the plastid envelope. Physiol. Plant 100,
McAndrew, R.S., Froehlich, J.E., Vitha, S., Stokes, K.D., and
Osteryoung, K.W. (2001). Colocalization of plastid division proteins
in the chloroplast stromal compartment establishes a new functional
relationship between FtsZ1 and FtsZ2 in higher plants. Plant Physiol.
Miege, C., Marechal, E., Shimojima, M., Awai, K., Block, M.A., Ohta,
H., Takamiya, K., Douce, R., and Joyard, J. (1999). Biochemical and
topological properties of type A MGDG synthase, a spinach chloro-
plast envelope enzyme catalyzing the synthesis of both prokaryotic
and eukaryotic MGDG. Eur. J. Biochem. 265, 990–1001.
Mongrand, S., Besoule, J.-J., Cabantous, F., and Cassagne, C.
(1998). The C16:3/C18:3 fatty acid balance in photosyntheitc tissues
from 468 plant species. Phytochemistry 49, 1049–1064.
Murashige, T., and Skoog, F. (1962). A revised medium for rapid
growth and bioassays with tobacco tissue cultures. Physiol. Plant 15,
Nishida, I., Tasaka, Y., Shiraishi, H., and Murata, N. (1993). The gene
and the RNA for the precursor to the plastid-located glycerol-3-
phosphate acyltransferase of Arabidopsis thaliana. Plant Mol. Biol. 21,
Ohlrogge, J., and Browse, J. (1995). Lipid biosynthesis. Plant Cell 7,
Ohnishi, J., and Yamada, M. (1982). Glycerolipid synthesis in Avena
leaves during greening of etiolated seedlings III. Synthesis of a
linolenoyl monogalactosyl diacylglycerol from liposomal linoleoyl-
phosphatidylcholine by Avena plastids in the presence of phospha-
tidylcholine-exchange protein. Plant Cell Physiol. 23, 767–773.
Park, J., Gu, Y., Lee, Y., Yang, Z., and Lee, Y. (2004). Phosphatidic
acid induces leaf cell death in Arabidopsis by activating the Rho-
related small G protein GTPase-mediated pathway of reactive oxygen
species generation. Plant Physiol. 134, 129–136.
Pearce, M.L., and Slabas, A.R. (1998). Phosphatidate phosphatase
from avocado (Persea americana): Purification, substrate specificity
and possible metabolic implications for the Kennedy pathway and cell
signaling in plants. Plant J. 14, 555–564.
Pierrugues, O., Brutesco, C., Oshiro, J., Gouy, M., Deveaux, Y.,
Carman, G.M., Thuriaux, P., and Kazmaier, M. (2001). Lipid phos-
phate phosphatases in Arabidopsis. Regulation of the AtLPP1 gene in
response to stress. J. Biol. Chem. 276, 20300–20308.
Potocky, M., Elias, M., Profotova, B., Novotna, Z., Valentova, O., and
Zarsky, V. (2003). Phosphatidic acid produced by phospholipase D is
required for tobacco pollen tube growth. Planta 217, 122–130.
Rossak, M., Scha ¨fer, A., Xu, N., Gage, D.A., and Benning, C. (1997).
deficient mutant of Rhodobacter sphaeroides inactivated in sqdC.
Arch. Biochem. Biophys. 340, 219–230.
Roughan, P.G., Holland, R., and Slack, C.R. (1980). The role of
chloroplasts and microsomal fractions in polar-lipid synthesis from
[1–14C]acetate by cell-free preparations from spinach (Spinacia
oleracea) leaves. Biochem. J. 188, 17–24.
Roughan, P.G., and Slack, C.R. (1982). Cellular organization of
glycerolipid metabolism. Annu. Rev. Plant Physiol. 33, 97–132.
Sakaki, T., Saito, K., Kawaguchi, A., Kondo, N., and Yamada, M.
(1990). Conversion of monogalactosyldiacylglycerols to triacylglycer-
ols in ozone-fumigated spinach leaves. Plant Physiol. 94, 766–772.
Schunemann, D., Borchert, S., Flu ¨gge, U.I., and Heldt, H.W. (1993).
ADP/ATP translocator from pea root plastids (comparison with trans-
locators from spinach chloroplasts and pea leaf mitochondria). Plant
Physiol. 103, 131–137.
Shimojima, M., Ohta, H., Iwamatsu, A., Masuda, T., Shioi, Y., and
Takamiya, K. (1997). Cloning of the gene for monogalactosyldiacyl-
glycerol synthase and its evolutionary origin. Proc. Natl. Acad. Sci.
USA 94, 333–337.
Silva-Filho, M.D., Wieers, M.C., Flu ¨gge, U.I., Chaumont, F., and
Boutry, M. (1997). Different in vitro and in vivo targeting properties of
the transit peptide of a chloroplast envelope inner membrane protein.
J. Biol. Chem. 272, 15264–15269.
Thiery, L., Leprince, A.S., Lefebvre, D., Ghars, M.A., Debarbieux, E.,
and Savoure, A. (2004). Phospholipase D is a negative regulator of
proline biosynthesis in Arabidopsis thaliana. J. Biol. Chem. 279,
Tranel, P.J., Froehlich, J., Goyal, A., and Keegstra, K. (1995). A
component of the chloroplastic protein import apparatus is targeted
to the outer envelope membrane via a novel pathway. EMBO J. 14,
Tranel, P.J., and Keegstra, K. (1996). A novel, bipartite transit peptide
targets OEP75 to the outer membrane of the chloroplastic envelope.
Plant Cell 8, 2093–2104.
Vitha, S., Froehlich, J.E., Koksharova, O., Pyke, K.A., van Erp, H.,
and Osteryoung, K.W. (2003). ARC6 is a J-domain plastid division
protein and an evolutionary descendant of the cyanobacterial cell
division protein Ftn2. Plant Cell 15, 1918–1933.
Voelker, T., and Kinney, A.J. (2001). Variations in the biosynthesis of
seed-storage lipids. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52,
Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D. (2003). An
enhanced transient expression system in plants based on suppres-
sion of gene silencing by the p19 protein of tomato bushy stunt virus.
Plant J. 33, 949–956.
Weber, A., Menzlaff, E., Arbinger, B., Gutensohn, M., Eckerskorn,
C., and Flu ¨gge, U.I. (1995). The 2-oxoglutarate/malate translocator of
chloroplast envelope membranes: molecular cloning of a transporter
containing a 12-helix motif and expression of the functional protein in
yeast cells. Biochemistry 34, 2621–2627.
Xu, C., Fan, J., Riekhof, W., Froehlich, J.E., and Benning, C. (2003). A
permease-like protein involved in ER to thylakoid lipid transfer in
Arabidopsis. EMBO J. 22, 2370–2379.
Xu, C., Ha ¨rtel, H., Wada, H., Hagio, M., Yu, B., Eakin, C., and
Benning, C. (2002). The pgp1 locus of Arabidopsis encodes a phos-
phatidylglycerol synthase with impaired activity. Plant Physiol. 129,
Zhang, W., Wang, C., Qin, C., Wood, T., Olafsdottir, G., Welti, R., and
Wang, X. (2003). The oleate-stimulated phospholipase D, PLDdelta,
and phosphatidic acid decrease H2O2-induced cell death in Arabi-
dopsis. Plant Cell 15, 2285–2295.
3110The Plant Cell
DOI 10.1105/tpc.105.035592 Download full-text
; originally published online September 30, 2005; 2005;17;3094-3110
Changcheng Xu, Jilian Fan, John E. Froehlich, Koichiro Awai and Christoph Benning
Mutation of the TGD1 Chloroplast Envelope Protein Affects Phosphatidate Metabolism in
This information is current as of May 27, 2014
This article cites 64 articles, 31 of which can be accessed free at:
Sign up for eTOCs at:
Sign up for CiteTrack Alerts at:
is available at:
The Plant Cell
Subscription Information for
ADVANCING THE SCIENCE OF PLANT BIOLOGY
© American Society of Plant Biologists