Tocopherols Modulate Extraplastidic Polyunsaturated Fatty
Acid Metabolism in Arabidopsis at Low Temperature
Hiroshi Maeda,a,b,1Tammy L. Sage,cGiorgis Isaac,dRuth Welti,dand Dean DellaPennaa,b,2
aDepartment of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824
bCell and Molecular Biology Program, Michigan State University, East Lansing, Michigan 48824
cDepartment of Ecology and Evolutionary Biology, University of Toronto, Toronto, Ontario, Canada M5S 3B2
dKansas Lipidomics Research Center, Division of Biology, Kansas State University, Manhattan, Kansas 66506
Tocopherols (vitamin E) are synthesized in plastids and have long been assumed to have essential functions restricted to
these organelles. We previously reported that the vitamin e-deficient2 (vte2) mutant of Arabidopsis thaliana is defective in
transfer cell wall development and photoassimilate transport at low temperature (LT). Here, we demonstrate that LT-treated
vte2 has a distinct composition of polyunsaturated fatty acids (PUFAs): lower levels of linolenic acid (18:3) and higher levels
of linoleic acid (18:2) compared with the wild type. Enhanced 18:3 oxidation was not involved, as indicated by the limited
differences in oxidized lipid species between LT-treated vte2 and the wild type and by a lack of impact on the LT-induced
vte2 phenotype in a vte2 fad3 fad7 fad8 quadruple mutant deficient in 18:3. PUFA changes in LT-treated vte2 occur primarily
in phospholipids due to reduced conversion of dienoic to trienoic fatty acids in the endoplasmic reticulum (ER) pathway.
Introduction of the ER fatty acid desaturase mutation, fad2, and to a lesser extent the plastidic fad6 mutation into the vte2
background suppressed the LT-induced vte2 phenotypes, including abnormal transfer cell wall development. These results
provide biochemical and genetic evidence that plastid-synthesized tocopherols modulate ER PUFA metabolism early in the
LT adaptation response of Arabidopsis.
for mammals and, together with tocotrienols, are collectively
known as vitamin E (Evans and Bishop, 1922; Bramley et al.,
2000; Schneider, 2005). Based on in vitro studies, these lipid-
soluble compounds are effective antioxidants that quench sin-
glet oxygen and scavenge lipid peroxyl radicals and hence
terminate the autocatalytic chain reaction of lipid peroxidation
(Tappel, 1972; Fahrenholtz et al., 1974; Burton and Ingold, 1981;
Liebler and Burr, 1992; Kamal-Eldin and Appelqvist, 1996).
Tocopherols localize in membranes where they associate with
polyunsaturated fatty acids (PUFAs) and affect properties such
as the permeability and stability of membranes (Erin et al., 1984;
Kagan, 1989; Stillwell et al., 1996; Wang and Quinn, 2000).
In animals, tocopherol deficiency has severe consequences,
including neurological dysfunction and muscular dystrophy
(Bramley et al., 2000; Schneider, 2005). Because tocopherol de-
ficiency symptoms are often associated with increased oxidative
stresses, it has been generally acceptedthat tocopherols primarily
function as antioxidants in animal membranes. Studies with mam-
malian cell cultures have led to the proposal that specific forms of
tocopherols (e.g., a-tocopherol but not g-tocopherol) also have
functions unrelated to their antioxidant properties, which include
modulation of signaling pathways (Pentland et al., 1992; Ricciarelli
et al., 1998, 2002; Jiang et al., 2000; Rimbach et al., 2002).
However, the underlying mechanisms of tocopherol function(s) in
animals remain an open question.
Despite the fact that tocopherols are synthesized only by
photosynthetic organisms, including all plants and algae and
some cyanobacteria, until recently there was no direct evidence
demonstrating the roles of tocopherols in photosynthetic organ-
isms (Fryer, 1992; Munne-Bosch and Alegre, 2002; Maeda and
DellaPenna, 2007). The tocopherol-deficient vte2 (for vitamin e2)
mutant of Arabidopsis thaliana is defective in homogentisate
phytyl transferase, the first committed enzyme of the tocopherol
biosynthetic pathway (Collakova and DellaPenna, 2001, 2003;
Savidge et al., 2002), and lacks all tocopherols and pathway
intermediates (Sattler et al., 2004). The vte2 mutant has reduced
seed viability and impaired seedling development, both of which
are associated with a massive elevation in lipid peroxidation
(Sattler et al., 2004, 2006), indicating that tocopherols play an
and early seedling development. However, mature vte2 plants
and the orthologous mutant in Synechocystis sp PCC6803
(Collakova and DellaPenna, 2001; Savidge et al., 2002) were
virtually indistinguishable from their respective wild types under
normal growth conditions and also during high intensity light
stress (Sattler et al., 2004; Maeda et al., 2006; Sakuragi et al.,
2006). They became distinguishable only under combinations of
1Current address: Department of Horticulture and Landscape Architec-
ture, Purdue University, 625 Agriculture Mall Dr., West Lafayette, IN
2Address correspondence to firstname.lastname@example.org.
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: Dean DellaPenna
WOnline version contains Web-only data.
The Plant Cell, Vol. 20: 452–470, February 2008, www.plantcell.org ª 2008 American Society of Plant Biologists
high light and low temperature or lipid peroxidation-inducing
chemical treatment (Havaux et al., 2005; Maeda et al., 2005,
high light stress than had been assumed.
In contrast with the lack of a visible phenotype in Arabidopsis
tocopherol-deficient mutantsunder normal growthandhigh light
stress conditions, tocopherol-deficient maize (Zea mays) and
potato (Solanum tuberosum) plants had reduced stature and
massive carbohydrate and anthocyanin accumulation in source
leaves, suggesting impairment of photoassimilate transport
(Russin et al., 1996; Provencher et al., 2001; Hofius et al., 2004).
Most recently, a similar carbohydrate and anthocyanin accumu-
lation phenotype was also observed in tocopherol-deficient
Arabidopsis vte2 mutants in response to nonfreezing low-
temperature (LT) treatment (i.e., 3 to 128C; Maeda et al., 2006).
As early as 6 h after transfer to LT, tocopherol deficiency in vte2
resulted in abnormal callose deposition and defective transfer
cell wall development in phloem parenchyma cells, either one or
both of which creates a bottleneck for photoassimilate export
from source tissues. The resulting inhibition of photoassimilate
export subsequently leads to carbohydrate and anthocyanin
accumulation after 3 and 14 d, respectively, and to growth inhi-
2006). Notably, the LT phenotypes of vte2 were independent
of light level and not associated with markers of photooxida-
tive stress (Maeda et al., 2006). These combined results from
tocopherol-deficient plants demonstrated a crucial role for to-
copherols in phloem loading for photoassimilate export.
Given the well-characterized chemical nature of tocopherols
as lipid-soluble antioxidants (Tappel, 1972; Liebler and Burr,
1992; Kamal-Eldin and Appelqvist, 1996) and their localization in
PUFA-enriched chloroplast membranes (Bucke, 1968; Soll etal.,
1985; Wise and Naylor, 1987), we hypothesized that tocopherol
deficiency in Arabidopsis impacts chloroplast membrane lipids
and that this might be an underlying cause of the impaired
phloem loading phenotype of tocopherol-deficient plants. In this
study, we conducted a detailed analysis of membrane lipid
oxidation, composition, and dynamics during LT treatment of
major impact on PUFA composition not due to elevated lipid
peroxidation in plastids but rather because of altered PUFA
synthesis in the endoplasmic reticulum (ER).
vte2 and the Wild Type Have Distinct Dienoic and Trienoic
Fatty Acid Levels at LT
To examine if tocopherol deficiency affects fatty acid composi-
tion of Arabidopsis leaves, vte2 and Columbia (Col) plants were
grown under permissive conditions (228C) for 4 weeks and the
middle portion of fully expanded rosette leaves harvested and
analyzed. The total fatty acid composition of Col and vte2 leaves
grown at permissive conditions was indistinguishable (Figure 1).
Because tocopherol deficiency has a dramatic impact on LT
adaptation of Arabidopsis (Maeda et al., 2006), we hypothesized
that tocopherol deficiency would affect membrane lipid compo-
sition at LT. To test this possibility, 4-week-old vte2 and Col
plants grown at 228C were transferred to LT (78C) conditions and
leaf total fatty acid compositions were compared after 14 d of LT
treatment. Col had higher a-linolenic acid (18:3) at 78C than it did
at 228C, as reported previously (Williams et al., 1988). Interest-
ingly, LT-treated vte2 had significantly higher linoleic acid (18:2)
and correspondingly lower 18:3 relative to Col (Figure 1).
PUFA Changes in LT-Treated vte2 Are Temporally
and Spatially Associated with Vascular-Specific
As previously reported, after transfer to LT, vte2 accumulated
callose initially in petiole vascular tissue and subsequently in an
acropetal fashion throughout leaf blade vascular tissue (Maeda
specific callose deposition is an early response of the vte2 LT-
induced phenotypes. To assess if LT-induced, vte2-dependent
PUFA alterations are related to vascular-specific callose depo-
sition, tissue from the petiole and mid-blade of mature leaves
was harvested after 0, 3, 7, and 14 d of LT treatment, and total
fatty acid composition and aniline blue–positive fluorescence, a
measure of callose deposition, were compared. Consistent with
the results from Figure 1, vte2 mid-blades in comparison to Col
showed lower 18:3 and higher 18:2 levels in response to LT and,
as a result, a significantly lower 18:3/18:2 ratio after 7 d of LT
treatment (Figure 2A; see Supplemental Table 1 online). In vte2
mid-blades, only a few weak aniline blue–positive fluorescence
spots were detected at 3 d, and their frequency and intensity
dramatically increased by 7 d (Figure 2B). No fluorescence was
detected in Col leaf blades and petioles at all time points (Maeda
et al., 2006). Thus, PUFA alterations appear temporally corre-
lated with callose deposition in vte2 leaf blades during LT
treatment. When 18:2 and 18:3 levels were analyzed from the
petioles of vte2 and Col plants, significant differences in 18:3/
18:2 ratios were observed even before LT treatment (Figure 2A),
whereas aniline blue–positive fluorescence in vte2 petioles first
became apparent after 6 h of LT treatment (Maeda et al., 2006)
Figure 1. Total Fatty Acid Composition of Col and vte2 Leaves before
and after 14 d of LT Treatment.
Col and vte2 plants were grown at 228C for 4 weeks, and the middle
portions of mature leaf blades were harvested for analysis before and
after 14 d of 78C treatment. Values for each fatty acid (x:y represents
number of carbons:number of unsaturated bonds) are means 6 SD (n ¼ 4
biological replicates) and are expressed as mol %.
Tocopherols Modulate ER Polyunsaturated Fatty Acid Metabolism 453
result indicates that vte2-specific PUFA alterations occur in
petioles prior to callose deposition and the onset of other vte2 LT
The vte2 Mutation Impacts PUFAs Derived from the ER
Pathway at LT
To further examine changes in membrane lipid composition in
after 14 d of LT treatment using electrospray ionization triple
quadrupole mass spectrometry (MS; Welti et al., 2002; Welti and
Wang, 2004; Devaiah et al., 2006). Under permissive conditions,
the levels of polar lipid classes (leaf 228C in Figure 3A) and the
composition of fatty acids esterified to each polar lipid class (leaf
228C in Figure 3B) were similar in vte2 and Col leaves, indicating
lipid and fatty acid composition of Arabidopsis leaves under
permissive growth conditions.
After 14 d of LT treatment, the head-group composition of
in Figure 3A), whereas the fatty acid composition of each polar
lipid class exhibited distinct changes especially in 18:3- and
18:2-containing acyl species (leaf 78C in Figure 3B). Digalacto-
syldiacylglycerol (DGDG) in vte2 relative to Col had higher 34:3
and lower 36:6 species, which mainly consist of pairs of 16:0 and
18:3 fatty acids (hereafter indicated as 16:0-18:3) and 18:3-18:3,
Welti et al., 2002). Monogalactosyldiacylglycerol (MGDG) in vte2
had lower 36:6 (18:3-18:3) and higher 34:6 (18:3-16:3) compared
with Col. Unexpectedly, the fatty acid composition of two major
phospholipids, phosphatidylcholine (PC) and phosphatidyl-
ethanolamine (PE), were strongly impacted by the vte2 mutation.
The mutant in comparison to Col had lower 36:6 (18:3-18:3) and
34:3 (16:0-18:3) and higher 36:4 (18:2-18:2) and 34:2 (16:0-18:2)
in both PC and PE (Figure 3B). These data were in accordance
with the decreased level of 18:3 in total fatty acid analysis of LT-
treated vte2 relative to Col (Figure 1).
The levels of individual lipid molecular species were also
analyzed from the petioles of 3-d LT-treated vte2 and Col plants.
As observed in 14-d LT-treated leaves, the composition of polar
lipid classes was similar between 3-d LT-treated vte2 and Col
petioles (petiole 78C in Figure 3A). The 36:6 (18:3-18:3) and 34:3
(16:0-18:3) acyl species of PC and PE were reduced, and 36:4
(18:2-18:2) and 34:2 (16:0-18:2) species were increased in the
unlike 14-d LT-treated leaves, the composition of fatty acids
esterified to MGDG and DGDG was similar between genotypes
in 3-d LT-treated petiole. These results indicate that the mo-
lecular species consistently decreased in the leaves and pet-
16:0-18:3 acyl pairs of PC and PE) are all derived from the ER
pathway (Figure 3C) and suggest that tocopherol deficiency
at LT primarily reduces the level of 18:3 derived from the ER
Figure 2. 18:3/18:2 Ratio and Callose Deposition in the Petioles and Leaf Blades of Col and vte2 during LT Treatment.
Col and vte2 plants were grown at 228C for 4 weeks and then transferred to 78C for an additional 14 d.
(A) Fatty acid composition of total lipid extracts was analyzed from the middle portions of leaf blades (left graph) and petioles (right graph) of Col (closed
circles) and vte2 (open squares). Data are means 6 SD (n ¼ 4 biological replicates). Asterisks represent significance levels using Student’s t test of vte2
relative to Col at each time point (* P < 0.05; ** P < 0.01).
(B) Aniline blue–positive fluorescence of vte2 leaf blades (left) and petioles (right) of vte2 after 0, 3, and 7 d of LT treatment. Bars ¼ 100 mm.
454The Plant Cell
Figure 3. Lipid Profiles of Col and vte2 Leaves and Petioles before and after 14 d of LT Treatment.
(A) and (B) Col and vte2 were grown at 228C for 4 weeks and then transferred to 78C. Black and white bars are Col and vte2 leaves, respectively, before
LT treatment. White-hatched and black-hatched bars are Col and vte2 leaves, respectively, after 14 d of LT treatment. White-dotted and black-dotted
bars are Col and vte2 petioles, respectively, after 3 d of LT treatment. Values are means 6 SD (n ¼ 5 biological replicates) and are expressed as mol %.
PI, phosphatidylinositol; PS, phosphatidylserine; PA, phosphatidic acid; FA, fatty acid.
(A) Mole percent of total polar lipid classes analyzed.
(B) Mole percent of fatty acids esterified to DGDG, MGDG, PC, and PE.
(C) A diagram summarizing membrane PUFA biosynthesis in Arabidopsis and the PUFA-containing lipid molecular species (e.g., PC and PE with 16:0
and 18:3 acyls shown at the right topcorner) that are higher (up arrows)and lower(down arrows) in LT-treated vte2 relative to Col. Black and gray arrows
indicate acyl pairs derived from the ER and plastid pathways, respectively, while the striped arrow indicates lipid species that can be produced from
both pathways. Dotted arrows illustrate proposed transfer of lipids between the ER and plastid.
Tocopherols Modulate ER Polyunsaturated Fatty Acid Metabolism455
Oxidized Membrane Lipid Species Are Not Elevated in
How does the absence of tocopherols in vte2 lead to lower 18:3
compared with Col at LT? Given the well-established roles of
tocopherols as lipid-soluble antioxidants and the massive in-
seedlings (Sattler et al., 2004, 2006), peroxidation of PUFAs,
especially 18:3, in LT-treated vte2 would seem an obvious ex-
planation. Though a prior report found no differences in the level
of lipid-soluble peroxides accumulated in LT-treated Col and
vte2 as detected by the ferrous oxidation xylenol orange assay
(FOX assay; Maeda et al., 2006), if the lipid peroxides in
LT-treated vte2 plants were rapidly converted to other oxylipins,
they would not be detectable by the FOX assay. To test this
possibility, total lipids were extracted from 3-d LT-treated vte2
and Col petioles, where the most rapid and obvious fatty acid
composition differences were observed between genotypes
(Figures 2 and 3), and oxygenated 18-carbon acyls esterified to
galactolipids (MGDG and DGDG) and phospholipids (PC, PE,
and phosphatidylglycerol [PG]) were analyzed.
Nominal masses that correspond nearly uniquely to oxidized
18-carbon acyl chains were identified using quadrupole time-of-
flight (Q-TOF; details in Methods and Supplemental Figure
1 online). These 18-carbon oxylipins include 18:2-O (18 carbons,
two double-bond equivalents, and one oxygen in addition to the
could correspond to hydroxy-octadienoic acid, 18:3-O to
hydroxy-octatrienoic acid or keto-octadienoic acid, and 18:2-
2O to hydroperoxy-octadienoic acid (Chechetkin et al., 2004;
Montillet et al., 2004). 18:4-O and 18:3-2O in wounded Arabi-
dopsis complex lipids have been implicated to be oxophyto-
et al., 2006), but could also correspond to keto-octatrienoic acid
(KOTE; structure illustrated in Ble ´e and Joyard, 1996) and hydro-
peroxy-octatrienoic acid, respectively. Anionic acyl fragment
masses corresponding to these oxygenated 18 carbon acyls
were identified on PC, PE, PG, MGDG, and DGDG molecular
species using triple quadrupole MS.
A total of 34 oxidized complex lipid molecular species were
of most oxidized complex lipid species were quite low and not
different between genotypes (Figure 4; see Supplemental Figure
1 online). Compared with samples subjected to more severe
stresses (e.g., wounding), the LT-treated petioles had much less
oxidized complex lipid (Buseman et al., 2006). Although this
analysis does not provide one-to-one identification between a
detected ion and an individual compound (e.g., 18:4-O could be
either OPDA or 13-KOTE), these results indicate that membrane
lipid oxidation is not occurring at significantly elevated or differ-
ent levels in LT-treated vte2 and Col and that the lower level of
In Vivo Conversion of ER-Derived Dienoic to Trienoic Fatty
Acids Is Reduced in LT-Treated vte2
Lipid profiling data and oxidized lipid analysis suggested that
tocopherol deficiency in vte2 impacts 18:3 synthesis in the ER
pathway at LT. To directly test this possibility, lipid biosynthesis
in vte2 and Col was examined under permissive and LT condi-
tions using radiolabel tracer experiments. To assess early im-
pacts of tocopherol deficiency on lipid metabolism during LT
treatment, Col and vte2leaves were labeled with [14C]-acetate at
228C for 2 h and then transferred to 78C (or kept at 228C for
controls). The redistribution of radioactivity into different fatty
(Morris, 1966; Browse et al., 1986b). After 2 h of labeling at 228C,
enoic, dienoic, and trienoic fatty acids (see initial time points in
Figure 5A). When kept at 228C, incorporation of radioactivity into
trienoic fatty acids gradually increased to nearly 70% of total
activity, and incorporation into saturated, monoenoic, and di-
terns were almost identical between Col and vte2 (Figure 5A).
When transferred to 78C, vte2 in comparison to Col incorpo-
rated less radioactivity into trienoic fatty acids and more into
dienoic fatty acids: after 192 h, vte2 in comparison to Col
incorporated 3.6% 6 1.7% less and 4.6% 6 1.3% more14C
into trienoic and dienoic fatty acids, respectively (P < 0.01, n ¼ 3;
Figure 5A). Incorporation patterns into saturated and monoenoic
fatty acids at 78C were similar between genotypes: vte2 in
comparison to Col incorporated 0.4% 6 0.5% and 0.5% 6
1.1% less14C into monoenoic and saturated fatty acids, respec-
tively, at 192 h (P > 0.05, n ¼ 3). In a separate set of experiments,
plants were treated at 78C for 3 d prior to labeling at 78C and the
redistribution of radioactivity was examined. Under these con-
ditions, vte2 incorporated even less radioactivity into trienoic
fattyacids and more into dienoicfatty acidsin comparison to Col
(13.0% less and 10.3% more, respectively; see Supplemental
Figure 2A online). These results indicate that tocopherol
Figure 4. Oxylipin-Containing Polar Lipid Species in 3-d LT-Treated Col
and vte2 Petioles.
Compounds containing 18-carbon oxygenated fatty acyl anions were
analyzed by mass spectrometry as described in Methods. White- and
black-dotted bars are Col and vte2 petioles, respectively, after 3 d of LT
treatment. Data include 34 molecular species (9 PC, 10 PE, 3 PG, 7
MGDG, and 5 DGDG) with oxygenated 18-carbon fatty acids; details
about the analyzed species are given in Supplemental Figure 1 online.
Values are means 6 SD (n ¼ 5 biological replicates). No significant
differences were observed in all cases between genotypes (Student’s t
test, P > 0.05).
456 The Plant Cell
deficiency negativelyimpacts thein vivoconversion of dienoicto
trienoic fatty acids at LT.
The redistribution of radioactivity into different polar lipid
classes was also examined by separating total lipid extracts of
labeled leaves by thin layer chromatography (TLC). The MGDG,
PC, and PE bands were recovered from the TLC plates and
transmethylated and separated into different fatty acid methyl
esters on argentation TLC. Col and vte2 showed very similar
labeling patterns into various lipid classes at both 22 and 78C
(Figure 5B). The labeling patterns into different fatty acids ester-
vte2 when maintained at 228C (Figure 6).
When transferred to 78C, vte2 and Col exhibited nearly iden-
tical labeling patterns of MGDG acyl species, but vte2 incorpo-
rated less radioactivity into trienoic fatty acids and more into
dienoic fatty acids esterified to PC and PE than did Col. For
example, after 192 h, vte2 incorporated 10.0% 6 3.0% less and
11.7% 6 3.7% more14C into trienoic and dienoic fatty acids of
PC, respectively (P < 0.01, n ¼ 3; Figure 6). Similar but more
extreme trends were observed when plants were first LT-treated
for 3 d before being labeled and maintained at 78C (see Supple-
mental Figure 2C online). These results are in good agreement
with steady state lipid composition analysis (Figure 3) and
indicate that tocopherol deficiency at LT negatively impacts the
in vivo conversion of dienoic to trienoic fatty acids esterified to
PCandPE, bothofwhichareproduced throughtheERpathway.
14CO2labeling allows one to pulse label and chase (in ambient
air) to follow the turnover of total fatty acids (Bonaventure et al.,
2004), while [14C]-acetate labeling does not. Col and vte2 plants
were labeled with a 30-min pulse of14CO2and then chased in air
for an additional 8 d. Leaf tissue was harvested at different time
points, total membrane lipids were extracted and transmethyl-
ated, and the amount of radioactivity incorporated into total fatty
acids was determined. The level of radioactivity in fatty acids
increased during the first 7 h after labeling and gradually de-
creased thereafter (Figure 7). The slope of reduction of radio-
activity in total fatty acids was similar between Col and vte2
(Figure 7) even when corrected for the dilution effect caused
by growth (see Supplemental Figure 3 online). These results
suggestthatCol andvte2turnover fattyacids atsimilarrates and
that tocopherol deficiency does not significantly affect this
fad2 and fad6, But Not fad3, fad7, or fad8, Suppress
the Impaired Photoassimilate Export Phenotype of
To further examine the relationship between PUFA alterations
and the LT-induced phenotype of vte2, a series of mutations
affecting plastid- or ER-localized fatty acid desaturases were
introduced into the vte2 background and the consequences for
the vte2 LT phenotypes were assessed. In higher plants, PUFAs
are synthesized through both prokaryotic (chloroplast) and eu-
karyotic (ER) pathways (Roughan et al., 1980; Browse et al.,
1986b; Figure 3C). FAD2 and FAD3 are ER-localized fatty acid
desaturases; thus, the fad2 and fad3 mutants have reduced
PUFA content predominantly in phospholipids, the major lipid
components of extraplastidic membranes (Miquel and Browse,
Figure 5. Redistribution of Radioactivity among the Fatty Acids and
Polar Lipids of Col and vte2 at 22 or 78C.
Col (closed symbols) and vte2 (open symbols) were grown at 228C for 4
weeks, labeled with [14C]-acetate at 228C at time zero, and harvested for
the first time point 2 h later, after which time the labeled plants were
either kept at 228C (left graphs) or transferred to 78C (right graphs) for the
indicated times. All experiments were repeated three times with similar
trends. Representative data are shown.
(A) Redistribution of radioactivity among individual fatty acids. dia-
monds, saturated; squares, monoenoic; triangles, dienoic; circles, tri-
enoic fatty acids.
(B) Redistribution of radioactivity among individual polar lipids. Top
panels: squares, MGDG; diamonds, PG; triangles, DGDG; circles, SQDG
(sulfoquinovosyldiacylglycerol). Bottom panels: circles, PC; diamonds,
PE; triangles, PI.
Tocopherols Modulate ER Polyunsaturated Fatty Acid Metabolism457
1992; Browse et al., 1993; Okuley et al., 1994). FAD6, FAD7, and
FAD8 are plastid-localized fatty acid desaturases, and the fad6
and fad7 fad8 mutants have reduced PUFAs predominantly in
galactolipids, the major lipid components of plastidic mem-
branes (Browse et al., 1989; Falcone et al., 1994; McConn et al.,
1994). FAD2 and FAD6 convert monoenoic- to dienoic-fatty
acids, whereas FAD3, FAD7, and FAD8 convert dienoic- to
trienoic fatty acids; thus, compared with Col, the fad2 and fad6
mutants contain higher 18:1 and the fad3, fad7, and fad8 have
higher 18:2 and all these genotypes have lower 18:3 (Browse
McConn et al., 1994; Okuley et al., 1994; Wallis and Browse,
2002). The fad3 fad7 fad8 triple mutant lacks all trienoic fatty
acids (i.e., 18:3 and 16:3; McConn and Browse, 1996).
Under permissive growth conditions, the visible phenotype
(see Supplemental Figure 4 online) and fatty acid composition
(Table 1) of all fad-containing vte2 mutant lines were visually
indistinguishable from the corresponding single, double, or triple
transferred to 78C conditions for an additional 4 weeks, the fad2,
fad6, fad3, fad7 fad8, and fad3 fad7 fad8 mutants were also
indistinguishable from Col (Figure 8A). Although the fad2, fad6,
do not induce chilling sensitivity in these mutants (Hugly and
Somerville, 1992;Miquel etal.,1993;Routaboul etal.,2000).The
vte2 mutant showed the expected LT-induced phenotype, and
vte2 fad3, vte2 fad7 fad8, and vte2 fad3 fad7 fad8 mutants
exhibited a visible phenotype similar to vte2 (Figure 8A).
Figure 7.14CO2Pulse Chase Labeling of Total Fatty Acids in LT-Treated
Col and vte2.
Col (closed circles) and vte2 (open squares) grown at 228C for 3 weeks
were transferred to 78C for 3 d and then pulse labeled with14CO2for 30
min and chased in air at 78C. At the indicated times, leaf samples were
harvested and the specific activity in total fatty acids determined.
Samples for the initial time point were taken immediately after labeling
(30 min). Values are means 6 SD (n ¼ 3 biological replicates) and are
expressed as radioactivity detected in total fatty acids per mg fresh
weight (FW). Estimated rates of fatty acid turnover are indicated as the
slopes of solid (Col) and dotted (vte2) lines based on values starting at the
24-h time point.
Figure 6. Redistribution of Radioactivity among Fatty Acids of Individual
Lipids from Col and vte2 at 22 or 78C.
Col (closed symbols) and vte2 (open symbols) were treated and labeled
as in Figure 5. Redistribution of radioactivity among individual fatty acid
methylesters of MGDG, PC, or PE was analyzed by TLC. All experiments
were repeated three times, except for 78C MGDG and PE, which were
repeated two times. All experiments showed similar trends, and repre-
sentative data are shown. For the 78C experiment, an additional exper-
iment was also conducted using 3-d LT-treated plants, and similar but
more obvious trends were observed (see Supplemental Figure 2 online).
Diamonds, saturated; squares, monoenoic; triangles, dienoic; circles,
trienoic fatty acids.
458The Plant Cell
Interestingly, vte2 fad2, and to a lesser extent vte2 fad6, sup-
pressed the visible phenotype (i.e., purple leaf coloration and
reduced plant size) of LT-treated vte2 (Figure 8A). The suppres-
sion of the plant size was even more obvious when 3-week-old
plants grown under permissive conditions were transferred to LT
(see Supplemental Figure 5 online).
To assess the biochemical consequences of introducing fad
mutations into the vte2 background, photoassimilate export
capacity and the levels of soluble sugars (i.e., sucrose, glucose,
and fructose) were analyzed in all genotypes after 7 and 14 d of
LT treatment, respectively. These are the time points when vte2
and Col first showed differences that were substantial enough to
readily detect intermediate levels for these parameters (Maeda
et al., 2006). The total soluble sugar content of fad2, fad6, fad3,
fad7 fad8, and fad3 fad7 fad8 was similar to Col (29.5 6 12.3
mmol/gFW) with the exception of a slightly elevated level in fad3
fad7 fad8 (Figure 9A). The total soluble sugar content of vte2
vte2 (256.6 6 39.6 mmol/gFW). In vte2 fad2, this trait was
suppressed to the level in Col, while vte2 fad6 showed partial
suppression and a soluble sugar level intermediate between Col
and vte2 (Figure 9A). The starch levels of vte2 fad2 and vte2 fad6
Figure 6 online). The photoassimilate export capacity of the
different genotypes was negatively correlated to their soluble
sugar levels (cf. Figures 9A and 9B). The export capacity of fad2,
fad6, fad3, fad7 fad8, and fad3 fad7 fad8 was similar to Col, with
the exception of a slightly lower level in fad3 (Figure 9B). In vte2,
vte2 fad3, vte2 fad7 fad8, and vte2 fad3 fad7 fad8, we observed
similar and dramatically reduced photoassimilate export capac-
ities compared with Col. Interestingly, vte2 fad2 fully recovered
export capacity back to the Col level, while vte2 fad6 showed
significantly higher export capacity than vte2, though still lower
than Col or vte2 fad2 (Figure 9B). These results indicate that the
introduction of fad2, and to a lesser extent fad6, but not fad3,
the impaired photoassimilate export and sugar accumulation
phenotypes of vte2. The results with vte2 fad3 fad7 fad8 also
indicate that complete elimination of trienoic fatty acids has no
impact on the vte2 LT-induced phenotype.
Having eliminated trienoic fatty acid levels as being involved in
induction of the vte2 LT phenotype, we further analyzed the total
fatty acid composition of all genotypes to assess whether one or
more other fatty acids might be specifically associated with the
vte2 LT response or its suppression. After 14 d of LT treatment,
vte2 showed reduced 18:3 and increased 18:2 levels relative to
not occur in the vte2 fad2 genotype, and instead 18:1 and 16:0
Table 1. Fatty Acid Composition of Indicated Genotypes before and after 14 d of LT Treatment
Before LT treatment
vte2 fad7 fad8
fad3 fad7 fad8
vte2 fad3 fad7 fad8
16.6 6 2.1
15.9 6 1.6
17.4 6 1.8
17.1 6 2.3
15.9 6 1.0
15.6 6 0.6
16.1 6 2.9
15.1 6 1.7
13.2 6 0.1
13.0 6 0.6
12.4 6 0.7
12.4 6 0.4
3.2 6 0.4
3.1 6 0.4
2.9 6 0.6
2.8 6 0.6
10.3 6 1.0
10.3 6 1.0
3.1 6 0.4
3.5 6 0.7
2.6 6 0.1
2.5 6 0.2
2.5 6 0.1
2.4 6 0.1
0.6 6 0.1
0.6 6 0.2
0.5 6 0.1
0.4 6 0.1
0 6 0
0 6 0
0.6 6 0.1
0.7 6 0.1
7.6 6 0.2
7.9 6 0.3
7.7 6 1.0
7.6 6 0.3
6.9 6 1.0
7.5 6 1.4
7.7 6 1.4
6.8 6 1.1
0 6 0
0 6 0
7.0 6 2.0
8.0 6 1.2
0 6 0
0 6 0
0 6 0
0 6 0
1.1 6 0.1
1.0 6 0.2
0.8 6 0.3
0.5 6 0.1
0.3 6 0.3
0.5 6 0.4
0.9 6 0.1
0.9 6 0.1
1.2 6 0.4
1.1 6 0.3
1.5 6 0.7
0.6 6 0.1
5.9 6 0.5
5.7 6 1.2
24.3 6 2.5
25.6 6 4.5
30.7 6 1.6
30.5 6 1.7
6.2 6 1.0
5.9 6 0.9
6.2 6 0.6
6.6 6 0.5
6.4 6 0.9
6.1 6 0.3
17.8 6 1.9
17.6 6 2.4
5.2 6 0.5
5.0 6 0.5
17.7 6 0.5
18.6 6 0.6*
24.7 6 4.3
24.3 6 2.5
55.3 6 1.7
55.1 6 0.6
69.5 6 1.3
70.8 6 0.5
48.0 6 3.5
48.4 6 4.3
41.3 6 2.6
41.9 6 1.9
25.1 6 2.3
24.5 6 2.2
41.3 6 6.4
41.7 6 4.0
13.9 6 1.4
13.8 6 1.2
0 6 0
0 6 0
After 14 d of LT treatment
vte2 fad7 fad8
fad3 fad7 fad8
vte2 fad3 fad7 fad8
15.9 6 1.8
17.3 6 1.4
23.8 6 1.7
20.4 ± 1.8*
19.8 6 0.5
19.3 6 0.6
16.4 6 1.0
20.5 6 3.1*
17.7 6 0.8
18.5 6 0.4
13.3 6 0.4
13.4 6 0.2
1.5 6 0.1
1.4 6 0.2
2.4 6 0.2
2.3 6 0.3
7.5 6 0.3
8.2 6 0.3**
1.3 6 0.1
1.0 ± 0.2*
1.7 6 0.1
1.5 6 0.1
1.5 6 0.1
1.2 ± 0.0**
0.4 6 0.2
0.4 6 0.1
0.3 6 0.0
0.2 ± 0.0**
0 6 0
0 6 0
0.2 6 0.0
0.2 ± 0.0*
5.8 6 0.5
5.6 6 0.1
6.0 6 0.4
6.2 6 0.3
6.4 6 0.5
6.0 6 0.7
3.7 6 1.2
2.4 6 1.1
0 6 0
0 6 0
6.8 6 0.5
4.6 ± 1.7*
0 6 0
0 6 0
0 6 0
0 6 0
1.2 6 0.8
1.2 6 0.3
0.8 6 0.1
0.6 6 0.2
0.6 6 0.0
0.6 6 0.0
0.4 6 0.1
0.6 6 0.1*
0.4 6 0.0
0.5 6 0.0**
0.9 6 0.4
0.5 6 0.2
4.0 6 0.4
4.1 6 0.4
16.8 6 2.0
24.0 6 2.2**
27.3 6 1.1
28.0 6 0.9
3.2 6 0.5
4.2 6 0.7*
6.1 6 0.9
6.1 6 1.0
6.2 6 0.6
6.1 6 0.4
18.2 6 0.7
23.7 6 1.1**
13.0 6 1.3
13.6 6 0.9
22.5 6 1.2
25.1 6 1.7*
28.0 6 2.4
31.9 6 3.0
54.1 6 2.7
59.9 6 0.8**
72.1 6 0.8
72.5 6 0.6
52.4 6 1.8
46.0 ± 2.5**
39.4 6 3.1
36.5 6 4.1
22.2 6 1.6
18.7 ± 1.7*
43.7 6 3.2
37.1 ± 5.2*
14.3 6 1.9
7.9 ± 0.5**
0 6 0
0 6 0
Plants were grown at 228C for 4 weeks and then transferred to 78C for an additional 14 d. Values are means 6 SD (n ¼ 4 or 5 biological replicates) from
the middle portion of mature leaves and are expressed as mol %. Italics and bold indicate fatty acids that are significantly higher and lower,
respectively, in vte2 or vte2 containing fad mutant lines relative to the corresponding Col, single, or multiple fad mutant parents (Student’s t test, *P <
0.05, **P < 0.01).
Tocopherols Modulate ER Polyunsaturated Fatty Acid Metabolism 459
Figure 8. Visible Phenotype and Callose Deposition of LT-Treated Col, vte2, and a Series of fad and vte2-Containing fad Mutants.
All genotypes were grown at 228C for 4 weeks and then transferred to 78C.
(A) Representative plants of the indicated genotypes after 4 weeks of LT treatment. Bar ¼ 2 cm.
(B) Aniline blue–positive fluorescence in the lower half of the leaves after 3 d of LT treatment. Bar ¼ 1 mm.
were increased and decreased, respectively, in comparison to
fad2. These 18:2 and 18:3 changes still occurred when vte2 was
present in the fad3, fad6, and fad7 fad8 background, with the
exception of 18:2 in vte2 fad3. These fatty acid changes were
almost completely attenuated when vte2 was present in the fad3
fad7 fad8 background (Table 1). These results indicate that spe-
cific changes in membrane fatty acid composition per se do not
correlate with the degree of suppression of the vte2 LT pheno-
type in the various vte2-containing fad mutant genotypes.
fad2 and to a Lesser Extent fad6 Suppress the Abnormal
Transfer Cell Wall Development of LT-Treated vte2
Previous studies have demonstrated that reductions in photo-
assimilate export in LT-treated vte2 coincide with abnormal cell
wall morphology and callose deposition exclusively in phloem
parenchyma transfer cells (Maeda et al., 2006). To test if intro-
duction of the fad mutations into the vte2 background also
affects the vascular specific callose deposition at LT, 0-, 3-, and
7-d LT-treated plants were harvested and aniline blue–positive
fluorescence was analyzed as a marker of callose deposition.
The vascular tissue of Col and all fad mutants (fad2, fad6, fad3,
fad7 fad8, and fad3 fad7 fad8) lacked aniline blue–positive
fluorescence at all time points (Figure 8B; see Supplemental
Figure 7 online). By contrast, vascular specific aniline blue–
positive fluorescence appeared at the base of vte2 leaves at 3 d
and spread through the entire leaf by 7 d (Figure 8B; see
Supplemental Figure 7 online; Maeda et al., 2006). vte2 fad3,
vte2 fad7 fad8, and vte2 fad3 fad7 fad8 showed the same
patterns of aniline blue–positive fluorescence as vte2. vte2 fad6
also showed similar patterns to vte2, though the development
and degree of fluorescence in vte2 fad6 was slightly slower and
lower, respectively, thanvte2at3d(Figure8B)but notat7d(see
Supplemental Figure 7 online). vte2 fad2 had a background level
of fluorescence at all time points (Figure 8B; see Supplemental
Figure 7 online). These results indicate that fad2 and to a much
lesser extent fad6 but not fad3, fad7 fad8, or fad3 fad7 fad8
suppress the vascular specific callose deposition observed in
We further compared the patterns of phloem parenchyma
transfer cell wall development and callose deposition among LT-
treated Col, vte2, and vte2 fad2 and vte2 fad6, the two vte2-
containing fad lines that suppressed the photoassimilate export
and callose deposition phenotypes of vte2. Paradermal serial
sections and cross sections were examined with the transmis-
sion electron microscope after 3 d of LT treatment. In Col,
nascent, papillate cell wall ingrowths commonly found in other
species (DeWitt et al., 1999; Schmidt et al., 2000; Talbot et al.,
2001, 2002, 2007; Offler et al., 2002; Vaughn et al., 2007) were
initiated along the primary wall and were spatially associated
with Golgi, ER,and endomembrane vesicles (Figure 10A). Trans-
fer cell wall maturation progressed as described by Talbot et al.
(2001); papillate cell wall ingrowths branched (Figure 10B) and
projections were initiated (Figure 10D) that also branched and
fused to subsequently give rise to a mature fenestrated cell wall
that was composed of both an electron-opaque inner region and
electron-translucent outer region (Figures 10E and 10F), typical
of transfer cell wall structure (Vaughn et al., 2007). The LT-
induced initiation and maturation oftransfer cell wallsin fad2 and
fad6 were similar to Col (see Supplemental Figure 8 online).
The initiation of transfer cell wall development in vte2 and vte2
fad6 was also similar to Col as indicated by the presence of
papillate cell wall ingrowths (Figure 11A; see Supplemental
Figure 8 online) that branched laterally (Figure 11B) and fused
(Figure 11C). However, unlike Col, developmental polarity was
absent in vte2, and papillae were initiated around the entire cell.
Figure 9. Soluble Sugar Content and Photoassimilate Export Capacity
of LT-Treated Col, vte2, and a Series of fad and vte2-Containing fad
All genotypes were grown at 228C for 4 weeks and then transferred to
78C. Nonsignificant groups are indicated by alphabetical order with a
being the highest (analysis of variance, P < 0.05).
(A) After 14 d of LT treatment, mature leaves of the indicated genotypes
were harvested at the end of the light cycle and analyzed for total soluble
sugar content (i.e., glucose, black; fructose, gray; sucrose, white). Values
are means 6 SD (n ¼ 4 or 5 biological replicates) and expressed as
percentage of the vte2 value (256.6 6 39.6 mmol/gFW).
(B) After 7 d of LT treatment, mature leaves of the indicated genotypes
were labeled with14CO2 in the middle of the day, and14C-labeled
photoassimilate exudation was analyzed as described (Maeda et al.,
2006). Data are means 6 SD (n ¼ 5 or 6 biological replicates) and
expressed as percentage of the Col value (18.1% 6 3.1%14C exudated
per total14C fixed).
Tocopherols Modulate ER Polyunsaturated Fatty Acid Metabolism461
Figure 10. Cellular Structure, Cell Wall Development, and Immunodetection of b-1,3-Glucan in Col and vte2 fad2 after 3 d of LT Treatment.
(A) to (F) Col.
(G) to (I) vte2 fad2.
(A) to (I) Immunodetection of b-1,3-glucan. Single black asterisks mark nascent transfer cell wall papillae ([A] and [G]). Double asterisks mark laterally
elongating nascent papillate ingrowth (B). Black arrow marks fused, elongated papillae (C). White asterisks mark spherical wall ingrowths that have
developed on preexisting wall ingrowths ([D] and [H]). White arrowheads mark Golgi-derived vesicles ([A], [D], [G], and [H]). Black arrowhead marks
positive immunodetection of b-1,3-glucan (F). c, companion cell; e, endoplasmic reticulum; g, Golgi; m, mitochondrion; p, plastid, t, transfer cell wall.
Bars ¼ 0.5 mm.
462 The Plant Cell
Figure 11. Cellular Structure, Cell Wall Development, and Immunodetection of b-1,3-Glucan in vte2 after 3 d of LT Treatment.
(A) to (H) vte2. g, Golgi; m, mitochondrion; n, nucleus; p, plastid. Bars ¼ 0.5 mm.
(A) and (F) to (H) Immunodetection of b-1,3-glucan.
(A) Single black asterisk marks nascent transfer cell wall papilla.
(B) Double asterisks label laterally elongating nascent papillate ingrowth.
(C) Black arrow marks fused, elongated papillae.
(C) and (D) White asterisks mark hypertrophied, deformed wall ingrowths that have developed on preexisting wall ingrowths during early (C) and later
stages (D) of development.
(D) White arrow marks two coalesced ingrowths with origins on opposite sides of the cell as indicated by double white arrowheads.
(E) White arrowheads mark swollen, misshapen Golgi-derived vesicles.
(F) to (H) Black arrowheads mark positive immunodetection of b-1,3-glucan following fusion of laterally elongated papilla (F), through to the
development of continuously enlarging tumor-like ingrowths marked by triple black asterisks ([G] and [H]).
Tocopherols Modulate ER Polyunsaturated Fatty Acid Metabolism 463
continuous development of hypertrophied, deformed ingrowths
on the preexisting ingrowths (Figure 11C) that subsequently
coalesced and occluded the cell (Figure 11D). Golgi-derived
vesicles associated with developing wall ingrowths of LT-treated
vte2 were noticeably swollen and misshapen (Figure 11E) com-
pared with those of Col(Figures 10Aand 10D). InLT-treated vte2
fad6, developing ingrowths were varied in structure and were
either unevenly thickened and lacking extensive branching,
grossly enlarged, or in some cases normal (see Supplemental
Figure 8 online). Interestingly, in vte2 fad2, both initiation and
development of transfer cell walls were completely normal and
proceeded as in Col (Figures 10G to 10I).
The nascent papillae of all LT-treated genotypes lacked epi-
topes recognized by monoclonal antibodies to b-1,3-glucan
(Figures 10A, 10G, and 11A; see Supplemental Figure 8 online).
Positive immunolocalization for b-1,3-glucan was absent (Fig-
ures 10E and 10I) to low (Figure 10F) in LT-treated Col and vte2
fad2 and, when present, was only observed at the later stages of
transfer cell wall maturation (Figure 10F). By contrast, in vte2,
positive immunolocalization for b-1,3-glucan was noted contin-
uously following the time of convergence of laterally elongated
papillae (Figure 11F) through to the development of tumor-like
ingrowths (Figures 11G and 11H). These observations indicate
that the vte2 mutant appears to have normal initiation of LT-
induced transfer cell walls but that their development and mat-
uration is abnormal, a process that is completely and partially
suppressed by introduction of the fad2 and fad6 mutations,
All plants produce and accumulate tocopherols, yet their func-
tions are only beginning to be understood in photosynthetic
organisms. Previous studies revealed that tocopherols play a
crucial role in phloem loading and carbohydrate metabolism in
maize, potato, and cold-treated Arabidopsis (Russin et al., 1996;
Provencher etal., 2001; Porfirova etal., 2002;Sattler et al.,2003;
Hofius et al., 2004; Maeda et al., 2006). Tocopherol-deficient
parenchyma transfer cell walls and inhibited photoassimilate
export as early as 6 h after LT treatment, and this is followed by
elevated carbohydrate accumulation and ultimately growth inhi-
bition (Maeda et al., 2006). Although these phenotypes were
shown to be independent of any photoprotective role of tocoph-
erols, the underlying mechanisms were unknown. This study
focused on the impacts of tocopherol deficiency on membrane
structure and dynamics in LT-treated Arabidopsis leaves to
further understand tocopherol functions at LT.
Prior to LT treatment, the fatty acid and lipid composition of
vte2 and the wild type were indistinguishable, with the exception
of petiole tissue. In response to LT treatment, vte2 (and vte1, a
second tocopherol-deficient locus; Sattler et al., 2003) showed
higher 18:2 and lower 18:3 levels than the wild type (Figure 1;
data not shown). Consistent with a prior study reporting no
detectable increase in lipid peroxides in LT-treated vte2 (Maeda
et al., 2006), MS-based analysis for lipids with oxidized 18-
carbon acyl chains showed that almost all were at low levels and
not significantly different between LT-treated vte2 and the wild
type (Figure 4). Analysis of jasmonic acid (JA), OPDA, and phy-
toprostanes, all 18:3-derived oxylipins (Howe and Schilmiller,
2002; Farmer et al., 2003; Mueller, 2004), also showed no obvi-
ous increase in LT-treated vte2 (see Supplemental Figure 9
online). These combined data indicate that enhanced 18:3
oxidation does not contribute to the reduction in 18:3 levels in
LT-treated vte2 relative to the wild type.
Given that the majority of tocopherols reside in plastids, we
anticipated that the 18:3 reduction in vte2 would occur primarily
in PUFAs and lipids derived from the chloroplast pathway.
However, lipidomics showed that the level of plastid-derived
18:3-containing lipid species were indistinguishable between
genotypes in 3-d LT-treated petioles and was higher rather than
contrast, 18:3-containing species derived from the ER pathway
were significantly reduced, and ER-derived 18:2-containing
species were correspondingly increased in 3-d LT-treated pet-
ioles (Figure 3B). These differences were persistent and further
derived 18:3-containing lipid species (e.g., 18:3-18:3 of PC)
are imported back into the chloroplast and could eventually
affect chloroplast membrane composition (Figure 3C; Roughan
tocopherol deficiency preferentially impacts ER, rather than
chloroplastic, lipid metabolism. The results from [14C]-acetate
labeling analysis during the initial 8 d of LT treatment are in good
agreement with steady state lipidomic analysis: less and more
14C label was incorporated into newly synthesized trienoic and
esterified to ER-synthesized PC and PE but not chloroplast-
synthesized MGDG (Figure 6; see Supplemental Figure 2 online).
Finally, the fatty acid turnover rate was similar between LT-
treated vte2 and the wild type (Figure 7). These combined results
allow us to conclude that the primary impact of tocopherol
deficiency on lipids at LT is not on oxidation or metabolism of
PUFAs in the chloroplast but rather on the conversion of dienoic
to trienoic fatty acids in lipids produced in the ER.
Genetic modification of plastidic or extraplastidic PUFA syn-
background not only supports this conclusion but also provides
further insights into the underlying mechanisms of the vte2 LT-
induced phenotypes. The disruption of trienoic fatty acid syn-
thesis in the ER or plastid pathways (i.e., in vte2 fad3 and vte2
fad7 fad8) or the complete elimination of trienoic fatty acid
synthesis in both compartments (i.e., in vte2 fad3 fad7 fad8) had
no impact on the LT-induced phenotypes of vte2 (Figures 8 and
9).Thiswas in sharp contrastwiththe full suppression of the vte2
seedling phenotype in vte2 fad3 fad7 fad8 (Me `ne-Saffrane ´ et al.,
2007). Although it has been speculated that tocopherols are
involved in the regulation of trienoic fatty acid–derived signals
(Munne-Bosch and Falk, 2004, 2007), these data conclusively
eliminate the possibility that such compounds (e.g., JA, OPDA,
dinor-OPDA, and phytoprostanes) play a role in the initiation or
development of the tocopherol-deficient vte2 LT phenotype.
Furthermore, because trienoic fatty acids (i.e., 18:3 and 16:3)
make up ;95% of the PUFAs esterified to MGDG and DGDG,
the most abundant lipids in the chloroplast (Browse et al., 1989;
464 The Plant Cell
Miquel and Browse, 1992), these genetic data together with the
delayed and relatively minor impact of vte2 on galactolipid
species during LT treatment (Figure 3B) indicate that changes
to galactolipids are not responsible for the vte2 LT phenotype. In
contrast with fad3, fad7, and fad8, the introduction of fad2 or
fad6, mutations that affect the conversion of monoenoic to
dienoic fatty acids in the ER and chloroplast, respectively, had
dramatic impacts on the vte2 LT-induced phenotypes: fad2
completely suppressed while fad6 partially suppressed nearly all
vte2 LT phenotypes (Figures 8to 11; seeSupplemental Figures5
to 8 online). Because fad2 and to a lesser extent fad6 primarily
affect the fatty acid composition of phospholipids (Browse et al.,
1989; Miquel and Browse, 1992), alterations in phospholipid acyl
chains by fad2 or fad6 mutations are likely involved in the
suppression of the vte2 LT phenotype, consistent with the rapid
and major impact of vte2 on phospholipid molecular species
during LT treatment (Figure 3B). These combined results provide
genetic and biochemical evidence that membrane lipid metab-
olism, especially ER lipid metabolism, plays a central role in the
vte2-dependent LT-induced phenotypes.
Extraplastidic Functions of Tocopherols in Plants
Because tocopherols are synthesized in plastids (Soll et al.,
1980, 1985; DellaPenna and Pogson, 2006), they have long been
assumed to have essential functions restricted to this organelle
(Fryer, 1992; Munne-Bosch and Alegre, 2002). However, recent
studies using tocopherol-deficient photosynthetic organisms
have brought such an assumption into question (Maeda and
DellaPenna, 2007). Tocopherol deficiency in Arabidopsis and
Synechocystis sp PCC6803 has surprisingly subtle impacts on
responses to high intensity light stress, which primarily causes
oxidative stress in the chloroplast (Dahnhardt et al., 2002;
Porfirova et al., 2002; Havaux et al., 2005; Maeda et al., 2005,
be partially compensated for by other antioxidants (e.g., carot-
2005, 2006). By contrast, evidence has been accumulating
suggesting that tocopherol deficiency impacts specific extrap-
lastidic processes. Using the Arabidopsis vte2 mutants, Sattler
et al. (2004, 2006) have shown that tocopherols are essential in
preventing oxidation of PUFAs in triacylglycerols in seed oil
bodies, organelles derived from the ER in which up to 40% of
seed tocopherols accumulate (Yamauchi and Matsushita, 1976;
Fisk et al., 2006; White et al., 2006). Transfer cell wall develop-
ment at the plasma membrane is also severely impacted during
LT treatment of Arabidopsis vte2 (Maeda et al., 2006) as is
plasmodesmatal structure in the maize tocopherol-deficient
sxd1 mutant (Russin et al., 1996; Provencher et al., 2001), which
is coincident with impaired photoassimilate export in both sys-
tocopherols have an extraplastidic function in ER lipid metabo-
lism that causes abnormal transfer cell wall development and
impaired photoassimilate export during LT adaptation of Arabi-
dopsis. The impaired photoassimilate export phenotype of other
tocopherol-deficient mutants, such as maize and potato sxd1
(Russin et al., 1996; Provencher et al., 2001; Hofius et al., 2004),
may also be associated with similar changes in ER lipid metab-
olism. Introducing the orthologous fad2 and fad6 mutations into
tocopherol-deficient maize or potato sxd1 backgrounds could
directly test this hypothesis.
How do plastid-synthesized tocopherols affect lipid metabo-
lism in extraplastidic membranes? It has been well documented
that fatty acids produced in the chloroplast are exported to the
ER and transported back to the chloroplast (Browse et al., 1986b;
Pollard and Ohlrogge, 1999). Recently, the ER membrane was
et al., 2006) that provide a physical connection between the
chloroplast envelope and ER membranes and could allow trans-
fer of lipids and tocopherols between these two compartments.
Given that tocopherols are synthesized in the chloroplast inner
envelope (Soll et al., 1980, 1985) but are equally abundant in the
inner and outer envelope membranes (Soll et al., 1985), outer
envelope-localized tocopherols may also be transported to
extraplastidic membranes or at least preferentially localize to
the plastid-associated membrane regions where they could
directly interact with and impact the ER membrane or enzymes
involved in ER PUFA and lipid metabolism. However, at this
point, we cannot exclude the possibility that other chloroplast-
While it is clear that alteration of ER-derived phospholipids is a
direct result of the tocopherol deficiency in LT-treated vte2, it is
less clear how this leads to aberrant transfer cell wall develop-
symplast and apoplast (Offler et al., 2002), and LT strongly
stimulates phloem parenchyma transfer cell wall development in
Arabidopsis (Figure 10) presumably to increase the plasma
membrane surface area and associated transporters to maintain
photoassimilate transport capacity at LT (Bonnemain et al.,
1991; Harrington et al., 1997; Weber et al., 1997; Bagnall et al.,
2000). The magnitude and rapidity of transfer cell wall develop-
ment at LT in Arabidopsis places an extreme demand on the
secretory endomembrane system and extraplastidic lipid me-
tabolism for deposition and maintenance of cell wall and plasma
of endomembrane system components (i.e., ER, Golgi, and
associated vesicles) permeate Arabidopsis phloem parenchyma
transfer cells (Figures 10 and 11). In LT-treated vte2, initiation of
wall papilla appears to occur normally (Figure 11A), but polarized
transfer cell wall maturation is disrupted (Figures 11D and 11E),
and accumulate callose (Figures 11G and 11H; Maeda et al.,
2006). Interestingly, the abnormal cell wall maturation in LT-
treated vte2 is associated with hypertrophied Golgi-derived
vesicles that appear to be involved in deposition of transfer cell
wall components (Figures 11B and 11E; Aubert et al., 1996).
Studies in animal systems have shown that in addition to regu-
lating vesicular, organellar, and cellular membrane biogenesis
and morphology, specific membrane PUFAs modulate vesicle
fusion primarily through their interactions with SNAREs, key
proteins for membrane trafficking in all eukaryotes including
plants (Darios and Davletov, 2006; Connell et al., 2007; Davletov
et al., 2007; Latham et al., 2007).
Based on our observation and these animal studies, we hy-
pothesize that alteration in extraplastidic lipid metabolism as a
result of tocopherol deficiency affects properties of Golgi-derived
Tocopherols Modulate ER Polyunsaturated Fatty Acid Metabolism465
vesicles, thereby disrupting secretory processes that function
in normal transfer cell wall maturation in Arabidopsis at LT.
The LT-induced transfer cell wall development of Arabidopsis
phloem parenchyma cells and the maturation phase-specific
defect observed in vte2 provide a model system to further dis-
sect the control of transfer cell wall deposition and the under-
lying mechanism of the extraplastidic functions of tocopherols
Plant Materials and Construction of Double and Triple
The fad2-1, fad3-2, fad6-1, and fad7-1 fad8-1 mutants were obtained
from the ABRC. The vte2-1 mutant has been previously isolated and
backcrossed three times (Sattler et al., 2004). All genotypes are in the Col
background. The vte2-1 fad2-1, vte2-1 fad3-2, vte2-1 fad6-1 double,
vte2-1 fad7-1 fad8-1 triple, and vte2-1 fad3-2 fad7-2 fad8-1 quadruple
mutants were selected from crosses of the respective single or double
mutant parents. F2 progeny homozygous for the vte2-1 mutation were
identified by HPLC based on their tocopherol deficiency (Sattler et al.,
2004) and confirmed by genotyping with a vte2-1 cleaved-amplified
polymorphic sequence marker, 59-TTTCACTGGCATCTTGGAGGTA-
ATG-39 and 59-AAGTGGCAACTGTTTGTAGTAGAAG-39, which gener-
ates a 632-bp PCR product with a SacI site for the vte2-1 allele. F2
progeny homozygous for the respective fad mutation were identified by
fatty acid methyl ester analysis using gas–liquid chromatography as
described previously (Browse et al., 1986a).
Growth Conditions and LT Treatment
Seeds were stratified for 4 to 7 d (48C), planted in an equal mixture of
vermiculite, perlite, and soil fertilized with 13 Hoagland solution (see
conditions: 12 h, 120 mmol photon m?2s?1light at 228C/12 h darkness at
188C with 70% relative humidity. Plants were watered every other day and
photon m?2s?1light/12 h darkness at 78C (6<38C).
Carbohydrate Analyses, Phloem Exudation Experiments, and
Leaf-soluble sugar content (i.e., glucose, fructose, and sucrose) was
quantified using an enzymatic assay previously described (Maeda et al.,
2006). Phloem exudation experiments were conducted according to
Maeda et al. (2006) except that 0.05 mCi of NaH14CO3were converted to
14CO2for each labeling experiment and 10 mM EDTA was used for
exudation buffer. Phloem exudates were collected after 5 h of exudation.
The photoassimilate export capacity was measured as a percentage of
radioactivity exudated per total radioactivity fixed. Callose deposition
was visualized by aniline blue–positive fluorescence as previously de-
scribed (Maeda et al., 2006).
The profiles of individual lipid molecular species were obtained by an
automated electrospray ionization tandem mass spectrometry approach
as described previously (Devaiah et al., 2006). The 7th to 10th oldest
leaves from 4-week-old plants grown under permissive conditions were
cold treated for the indicated time, collected, and immediately immersed
in 758C isopropanol containing 0.01% butylated hydroxytoluene (BHT).
Total lipids were extracted as described by Welti et al. (2002) and
dissolved in chloroform for analysis.
Oxidized Lipid Analysis
Based on Q-TOF analysis, the acyl anions corresponding to 18-carbon
and C18H31O3(m/z 295), were designated as 18:4-O, 18:3-O, and 18:2-O
species, respectively, with the abbreviations indicating the number of
carbon atoms, the number of double bond equivalents that include C¼C,
C¼O,orringformationand thenumberofoxygenatoms inaddition tothe
indicated that these oxidized species, 18:4-O, 18:3-O, and 18:2-O, are
the major and generally the only lipid fragments with nominal m/z of 291,
293, and 295 (Buseman et al., 2006; Esch et al., 2007). The level of
oxylipin-containing lipid molecular species was quantified by precursor
scanning of oxylipin acyl anions, m/z 291, 293, and 295, using a triple
quadrupole mass spectrometer (API 4000; Applied Biosystems) in the
negative mode. The lipid extracts from 3-d LT-treated petioles were
dissolved in chloroform/methanol/aqueous 300 mM ammonium acetate
[M – H]?, PC species were identified as [M þ OAc]?, and MGDG and
DGDG species were identified as either [M – H]?or as [M þ OAc]?(see
Supplemental Figure 1 online). Product (Q-TOF) and precursor (triple
quadrupole) scanning demonstrated that product ion fragments corre-
spond either to entire acyl anions or to an acyl anion that has lost a water
molecule during collision-induced dissociation; i.e., in some cases, m/z
291 and 293 species have undergone a loss of water from m/z 309,
C18H29O4(18:3-2O) and m/z 311, C18H31O4(18:2-2O), respectively (see
Supplemental Figure 1 online). Although it is conceivable that oxylipins of
higher masses could produce m/z 291, 293, or 295 fragments through
additional water losses, examination of the detected precursor ion
species indicated that pairing m/z 291, 293, 295, 309, and 311 with
common and expected acyl species accounted for most of the spectral
peaks observed (see Supplemental Figure 1 online). Baselines were
subtracted, data were smoothed, and the centroids of a mass spectral
peak were determined (Analyst software; Applied Biosystems; for spec-
tral display only, peak intensities within one mass unit were binned). For
quantification, peak intensities were corrected for isotopic overlap from
nearby peaks. Mass spectral signals were normalized to the signal for
230 pmol di24:1 PG, an unnaturally occurring lipid species that was
added to the portion of the sample being analyzed as an internal
massspectralsignalfordi24 : 1PG3fractionof
Although no corrections for varying mass spectral response to the
various molecular species have been applied, these data provide for
direct comparison of the relative amounts of each molecular species in
wild-type plants compared with mutant plants.
Lipid Labeling Experiments
The 7th to 10th oldest leaves from 4-week-old plants grown under per-
missive conditions were labeled with 10 mL of 50 mCi/mL sodium [1-14C]-
acetate by applying 0.5 to 1 mL droplets to the leaf surface (Browse et al.,
1986b; Xu et al., 2003). Four labeled leaves were harvested into liquid
nitrogen from two plants after 2, 24, 72, 120, and 192 h of labeling.14CO2
pulse labeling experiments were conducted in a tightly sealed 10-liter
glass chamber as described (Maeda et al., 2006) using 2- to 3-week-old
plants and 0.5 mCi of NaH14CO3for acid-catalyzed production of14CO2.
466The Plant Cell
Total lipids were extracted in hot isopropanol as described (Hara and
Radin, 1978) and separated on 0.15 M ammonium sulfate-impregnated
TLC plates (Whatman K6 silica gel 60 A˚) using 91/30/8 (v/v/v) acetone/
toluene/water with 0.01% BHT as a solvent system. The TLC plates were
sprayed with 0.1% dichlorofluorescein (Sigma-Aldrich) to locate individ-
recovered and directly used for transesterification using 1 N hydrochloric
acid-methanol (Supelco; Browse et al., 1986a). The resulting fatty acid
methyl esters were separated based on the numbers of double bonds by
argentation (silver-coated) TLC (Morris, 1966). TLC plates were impreg-
nated with 10% (w/v) AgNO3in acetonitrile, activated for 5 min at 1008C,
developed three quarters of the way in 50% (v/v) and fully in 10% (v/v)
diethyl ether in hexane with 0.01% BHT. The radioactivity in each band
was quantified byexposing theplates to a phosphor screen togetherwith
standards with known radioactivity (Storm; GE Healthcare). Because the
30-min labeling time with14CO2will not saturate the lipid pool with14C,
the rate of fatty acid turnover calculated may be underestimated.
Transmission Electron Microscopy
Leaves were harvested in the middle of the third day after transfer to LT
(54 h of LT treatment) and prepared for transmission electron microscopy
and immunolocalization of b-1,3-glucan as described by Maeda et al.
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: Arabidopsis Vte1 (At4g32770/AF302188), Vte2 (At2g18950/
AF324344), Fad2 (At3g12120/NM112047), Fad3 (At2g29980/NM128552),
Fad6 (At4g30950/NM119243), Fad7 (At3g11170/NM111953), and Fad8
(At5g05580/NM120640). Seed information for the Arabidopsis mutants
used in this study can be found in The Arabidopsis Information Resource:
fad2-1 (CS8041), fad3-2 (CS8034), fad6-1 (CS207), and fad7-1 fad8-1
(CS8036). Seeds of double, triple, and quadruple mutants in the vte2
background do not germinate after a few months of storage (see Sattler
et al., 2004) and thus are maintained in the authors’ lab and are available
The following materials are available in the online version of this article.
Supplemental Figure 1. Individual Oxylipin-Containing Lipid Species
Analyzed in Figure 4.
Supplemental Figure 2. Redistribution of Radioactivity among the
Fatty Acids of Total Lipids or Individual Lipids of LT-Treated Col and
Supplemental Figure 3.14CO2Pulse Chase Labeling of Total Fatty
Acids in LT-Treated Col and vte2.
Supplemental Figure 4. Visible Phenotype before LT Treatment of
Col, vte2, and a Series of fad and vte2-Containing fad Mutants.
Supplemental Figure 5. Visible Phenotype of LT-Treated Col, vte2,
fad2, fad6, vte2 fad2, and vte2 fad6.
Supplemental Figure 6. Starch Content of LT-Treated Col, vte2,
fad2, fad6, vte2 fad2, and vte2 fad6.
Supplemental Figure 7. Callose Deposition of Col, vte2, and a Series
of fad and vte2-Containing fad Mutants after Prolonged LT Treatment.
Supplemental Figure 8. Cellular Structure, Cell Wall Development,
and Immunodetection of b-1,3-Glucan in fad2, fad6, and vte2 fad6
after 3 d of LT Treatment.
Supplemental Figure 9. The Level of JA, OPDA, and Phytoprostane
in Col and vte2-1 during LT Treatment.
Supplemental Table 1. Fatty Acid Composition of Total Lipid
Extracts from vte2 and Col Leaves and Petioles during a 14-d Time
Course of LT Treatment.
Supplemental Methods 1. The Composition of the Hoagland Solu-
tion Used in This Study.
We thank Kathy Sault for technical assistance with microscopy, John
Ohlrogge and Philip Bates for advice in labeled lipid analyses, Edward E.
Farmer and Laurent Me `ne-Saffrane ´ for the vte2 fad3 fad7 fad8 quadru-
ple mutant, Scott Sattler for the vte2 fad3 double mutant, William Pasutti
for assistance in selection of the vte2 fad6 double mutant, Ethan
Baughman, Pamela Tamura, and Mary Roth for Q-TOF analysis of
Arabidopsis lipid species, Martin Mueller for phytoprostane analysis,
and members of the DellaPenna lab for their critical advice, discussions,
and manuscript review. The Kansas Lipidomics Research Center Ana-
lytical Laboratory was supported by grants from the National Science
Foundation (MCB-0455318 and DBI-0521587) and the National Science
Foundation’s Experimental Program to Stimulate Competitive Research
(EPS-0236913), with matching support from the state of Kansas through
Kansas Technology Enterprise Corporation and Kansas State Univer-
sity, as well as from National Institutes of Health Grant P20 RR016475
from the IDeA Network of Biomedical Research Excellence program of
the National Center for Research Resources. This work was supported
by a Michigan State University strategic partnership grant and National
Science Foundation Grant MCB-023529 to D.D. and a Connaught
Award and Natural Sciences and Engineering Research Council of
Canada Discovery Grant to T.L.S.
Received July 31, 2007; revised January 4, 2008; accepted February 9,
2008; published February 26, 2008.
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