1. Seed Fatty Acid Reducer acts downstream of gibberellin
signalling pathway to lower seed fatty acid storage
in Arabidopsispce_2546 2155..2169
MINGXUN CHEN1
, XUE DU1
, YANG ZHU1
, ZHONG WANG1
, SHUIJIAN HUA3
, ZHILAN LI1,4
, WANGLI GUO1
,
GUOPING ZHANG1
, JINRONG PENG2
& LIXI JIANG1
1
Key Laboratory of Crop Germplasm Resource of Zhejiang Province, College of Agriculture and Biotechnology, 2
College of
Animal Sciences, Zhejiang University, Hangzhou 310058, 3
Zhejiang Academy of Agricultural Sciences, Hangzhou 310021,
and 4
Zhejiang Provincial Natural Science Foundation of China, Hangzhou 310012, China
ABSTRACT
Previous studies based on microarray analysis have found
that DELLAs down-regulate several GDSL genes in
unopened flowers and/or imbibed seeds. This suggests the
role of DELLAs in seed fatty acid (FA) metabolism. In the
present study, enhancement of gibberellin (GA) signalling
through DELLA mutation or exogenous gibberellin acid
A3 (GA3) resulted in the up-regulated expression of tran-
scription factors for embryogenesis and seed development,
genes involved in the FA biosynthesis pathway, and five
GDSL-type Seed Fatty Acid Reducer (SFAR) genes. SFAR
overexpression reduced the total seed FA content and led
to a particular pattern of seed FA composition. This ‘SFAR
footprint’ can also be found in plants with enhanced GA3
signalling. By contrast, the loss of SFAR function dramati-
cally increases the seed FA content. The transgenic lines
that overexpress SFAR were less sensitive to stressful envi-
ronments, reflected by a higher germination rate and better
seedling establishment compared with the wild type (WT)
plants. The GDSL-type hydrolyzer is a family of proteins
largely uncharacterized in Arabidopsis. Their biological
function remains poorly understood. SFAR reduces seed
FA storage and acts downstream of the GA signalling
pathway. We provide the first evidence that some GDSL
proteins are somehow involved in FA degradation in
Arabidopsis seeds.
Key-words: fatty acid; GA signalling pathway; GDSL-type
SFAR genes.
INTRODUCTION
Gibberellins (GAs) are a large group of tetracyclic diterpe-
noids that are essential for many aspects of plant growth
and development, such as seed germination, stem elonga-
tion, leaf expansion, trichome development, and flower and
fruit development (Swain, Reid & Kamiya 1997; King,
Moritz & Harberd 2001; Sasaki et al. 2003; Cheng et al. 2004;
Sun et al. 2004; Fleet & Sun 2005).The GA signal is received
and transduced by the GID1 GA receptor/DELLA repres-
sor pathway (Ueguchi-Tanaka et al. 2007). In Arabidopsis,
the DELLA proteins, namely, GA INSENSITIVE (GAI),
REPRESSOR OF ga1–3 (RGA), RGA-LIKE1 (RGL1),
RGA-LIKE2 (RGL2) and RGA-LIKE3 (RGL3), consti-
tute the nuclear negative regulators in the GA signalling
pathway (Peng & Harberd 1997; Silverstone, Ciampaglio &
Sun 1998; Yu et al. 2004). These five DELLA proteins have
both unique and overlapping functions (Silverstone et al.
2001; Lee et al. 2002; Jiang & Fu 2007). Genetic studies
indicate that GAI and RGA function in stem elongation as
GA-sensitive repressors (Peng & Harberd 1997; Silverstone
et al. 1998; Dill & Sun 2001). The loss of function of GAI
and RGA completely restores the dwarf phenotype,and the
combination of RGA, RGL1 and RGL2 loss-of-function
mutations represses petal, stamen filament and anther
development in ga1–3 mutants (Cheng et al. 2004; Yu et al.
2004). The RGL2 gene encodes the predominant repressor
of seed germination in Arabidopsis, and its function is
enhanced by the other DELLA proteins GAI, RGA and
RGL1 (Tyler et al. 2004; Cao et al. 2005).
Previous studies have indicated that GA regulates
embryogenesis during the torpedo and early cotyledon
stages when cells in the embryonic axis elongate (Hays,
Yeung & Pharis 2002). More recently, Singh et al. (2010)
reported that overexpression of a GA inactivation gene
causes seed abortion, demonstrating that active GAs in the
endosperm are essential for normal seed development.Gib-
berellin A3 (GA3) increases the unsaturation of fatty acid
(FA) in barley aleuronic layers, and this response exacer-
bates the disruption of endoplasmic reticulum function
under heat shock (Grindstaff, Fielding & Brodl 1996). The
application of exogenous GA3 to ga1–3 seedlings results in
drastic changes in the transcription of WRINKLED1
(WRI1), a central regulator of FA synthesis (Zentella et al.
2007). Less attention has been focused on the role of
DELLAs in regulating the positive and negative down-
stream factors that determine the final FA storage of a seed.
Correspondence: L. Jiang. e-mail: jianglx@zju.edu.cn; J. Peng.
e-mail: pengjr@zju.edu.cn
Plant, Cell and Environment (2012) 35, 2155–2169 doi: 10.1111/j.1365-3040.2012.02546.x
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2. Based on the result of microarray analysis which indi-
cated that DELLA proteins down-regulate several GDSL-
type genes in imbibed seeds, as well as in young Arabidopsis
flower buds (Cao et al. 2006), GA signals may modulate
seed FA metabolism. This is because GDSL-type proteins,
which have a serine-containing GDSL motif close to the
N-terminus, five conserved blocks (I to V), four strictly con-
served residues (Ser, Gly,Asn, and His in blocks I, II, III and
V, respectively) and a Ser-Asp-His triad in the amino acid
sequences, are active in the hydrolysis and synthesis of
lipids and esters (Beisson et al. 1997). This class of enzymes
is widely present in microbe and plant species. Important
members of this class include Aeromonas hydrophilia
lipases/acyltransferase, Vibrio parahaemolyticus hemolysin/
phospholipase, Xenorhabdus luminescens lipase, Brassica
napus proline-rich protein, Vibrio mimicus arylesterase and
Streptomyces rimosus lipase (Akoh et al. 2004). Studies
have shown that GDSL hydrolases have a flexible active
site that appears to change conformation with the presence
and binding of different substrates. Physiologically, plant
GDSL-type genes are mainly involved in the regulation of
plant growth and development. Up to 108 GDSL-type
genes are present in Arabidopsis, which display remarkable
structural diversity, with intron numbers ranging from 0 to
13. The genes, some of which are arranged in tandem, are
asymmetrically distributed in chromosomes 1 and 5 (Ling
2008). Presently, plant GDSL-motif enzymes have not been
proven to have any lipase activity. The Arabidopsis GDSL
LIPASE-LIKE 1 (GLIP1) is involved in ethylene signalling
and may mediate the production of systemic signalling mol-
ecules (Kwon et al. 2009), whereas GLIP2 plays a role in
resistance against Erwinia carotovora by negative regula-
tion of auxin signalling (Lee et al. 2009). Overexpression
of Arabidopsis thaliana LTL1, a salt-induced gene that
encodes a GDSL motif, increases salt tolerance in yeast and
transgenic plants (Naranjo et al. 2006).At present, there is a
paucity of information regarding the upstream regulators of
the GDSL-type genes and their role in determining seed FA
storage and cellular signalling.
In Arabidopsis, the total seed oil content among different
accessions varies widely from 33 to 43%. In contrast, the FA
composition is much more conservative (O’Neill et al.
2003). In plant, FA synthesis starts with the formation of
malonyl-CoA from acetyl-CoA, which is catalyzed by
acetyl-CoA carboxylase (ACCase). FA synthases catalyze
the transfer of the malonyl moiety to the acyl carrier
protein (ACP) by adding two carbons to the growing chain.
This leads to the formation of C16:0-ACPs and C18:0-
ACPs, which further elongate and unsaturate to form
various FA derivatives from the acyl chains. These nascent
FAs are then transported into the cytoplasm for lipid pro-
duction (Harwood 1996; Mu et al. 2008; Baud & Lepiniec
2009). ACCase is an important enzyme that controls the
initial step of FA synthesis. At least 24 enzymes and sub-
units are involved in the pathway. The genes that encode
most of the enzymes have been characterized and cloned
in Arabidopsis (Baud & Lepiniec 2009). FA biosynthesis
is regulated by several key transcription factors (TFs),
including WRI1, LEAFY COTYLEDON1 (LEC1), LEC2,
ABSCISIC ACID INSENSITIVE3 (ABI3) and FUSCA3
(FUS3).The WRI1 gene encodes a transcriptional regulator
of the AP2/EREB family that targets enzymes involved in
late glycolysis and in the plastidial FA biosynthetic network
(Focks & Benning 1998;Cernac & Benning 2004;Baud et al.
2009). Both LEC1 and LEC2 function as positive regula-
tors upstream of WRI1, ABI3 and FUS3, which jointly help
control the expression of seed storage proteins and the
genes related to FA (Kroj et al. 2003; Kagaya et al. 2005; Mu
et al. 2008; Pan et al. 2010). All these TFs have been shown
to participate in a regulatory cascade that controls the
expression of genes involved in seed FA biosynthesis during
seed maturation.
The present study investigates genes important for seed
development and FA metabolism among Arabidopsis with
reduced DELLA function to examine the activity of five
GDSL-type SFAR genes and their effect on seed FA
storage.
MATERIALS AND METHODS
Plant materials and growth conditions
The Arabidopsis ecotype Columbia (Col-0) was used for
the GA3 application and transformation studies. Lands-
berg erecta (Ler) was used for the generation of Q/ga1–3
(ga1–3, gai-t6, rga-t2, rgl1-1, and rgl2-1) and Q/WT (gai-t6,
rga-t2, rgl1-1 and rgl2-1) mutants as well as GA3 applica-
tion studies. The T-DNA insertion mutants (SALK
mutants) obtained from the Arabidopsis Biological
Resources Center were all on the Col-0 background. The
SALK mutants that mutated at loci SFAR1 (At1g54790),
SFAR2 (At1g58430), SFAR3 (At2g42990), SFAR4
(Ag3g48460) and SFAR5 (At4g18970) were ordered. They
were sfar1-1 (SALK_022225C), sfar1-2 (SALK_111581C),
sfar1-3 (SALK_063590), sfar2-1 (SALK_075941),
sfar3-1 (SALK_083121), sfar3-2 (SALK_123457), sfar4-1
(SALK_075665), sfar4-2 (SALK_008418C), sfar5-1,
(SALK_029865) and sfar5-2 (SALK_089605). The struc-
tures of the five SFAR genes and the locations of T-DNA
insertions of the SALK mutants are illustrated in
Supporting Information Fig. S1a. All T-DNA insertion
SALK mutants were backcrossed thrice with Col-0. The
homozygous T-DNA insertion lines were selected by
PCR genotyping (Supporting Information Fig. S1b) and
RT-PCR confirmation for null transcript of the respective
SFAR gene (Supporting Information Fig. S1c). All the
primers used for the PCR and RT-PCR analysis are
listed in Supporting Information Tables S1 and S2. The
purified homozygous mutants were used to analyse the
seed FA content and produced the nine double mutants:
sfar2-1 sfar3-2, sfar2-1 sfar4-1, sfar2-1 sfar5-1, sfar1-3
sfar3-1, sfar1-1 sfar4-1, sfar1-3 sfar5-1, sfar3-1 sfar4-2,
sfar3-1 sfar5-1 and sfar4-2 sfar5-1 by crossing and subse-
quent PCR screening. The growing conditions for Arabi-
dopsis were as our previous descriptions (Cao et al. 2005,
2006).
2156 M. Chen et al.
© 2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 2155–2169

3. Plasmid construction and plant transformation
The primers used to clone the cDNA of the five SFARs are
listed in Supporting Information Table S3. With SFAR2 as
an example, a 1.309 Kb SFAR2 cDNA fragment amplified
through PCR using KAPAHiFi™ DNA Polymerase was
cloned into modified pCAMBIA1300 driven by 35S. These
constructs were verified by extensive restriction endonu-
clease digestion and DNA sequencing and were then trans-
formed into Agrobacterium tumefaciens strain GV3101,
which was then used for the transformation of Arabidopsis
WT (Col-0) plants via floral dip (Clough & Bent 1998).The
transgenic plants were selected on solid Murashige and
Skoog (MS) medium with 30 mg mL-1
hygromycin and veri-
fied by PCR genotyping. T4 transgenic plants were gener-
ated and then RT-quantitative PCR was applied to verify
the transgenic plants further.
Application of exogenous GA3 to plants
Distilled water, used as the control, was set as Level-1
(0 mm) and a 500 mm GA3 solution was set as Level-2. The
different levels of GA3 solution were applied to eight indi-
vidual plants (Col-0) in one of three randomly arranged
blocks. At the bolting stage, the whole plants were sprayed
with 0 and 500 mm every other day until the first silique was
harvested. Traits, such as plant height, branch number,
number and length of siliques, and seed appearance, were
then recorded.
Seed morphology and oil bodies in
developing seeds
The seeds for observation were selected from the siliques
harvested from the basal part of a major inflorescence. Six
seeds of each genotype (or treatment) were randomly
selected for sectioning.Perpendicular transections were pro-
duced and the sections with the largest oval-shaped surface
area were selected for quantification.Twenty cells that locate
in the middle of a section were selected to count the oil
bodies within a cell.The seeds were photographed using an
OLYMPUS SZ 61 stereomicroscope (Tokyo, Japan). The
observation of the oil bodies under transmission electron
microscopy (TEM) (JEM-1230, Tokyo Japan) was per-
formed following the description by Eastmond (2006).
Analysis of FAs
FA extraction and analysis were carried out following Mu
et al. (2008). About 10 mg mature seeds were prepared by
heating intact seeds at 80 °C in a methanol solution con-
taining 1 m HCl for 2 h. The FA methyl esters were
extracted with 2 mL hexane and 2 mL 0.9% (w/v) NaCl,
and the organic phase was used for analysis by gas chroma-
tography (GC), using methyl heptadecanoate as an internal
standard. The machine (SHIMADZU, Kyoto, Japan,
GC-2014) was equipped with a flame ionization detector
and a 30 m (length) ¥ 0.25 mm (inner diameter) ¥ 0.5 mm
(liquid membrane thickness) column (Supelco wax-10,
Supelco, Cat. no. 24079, Schnelldorf, Germany). The initial
column temperature was maintained at 160 °C for 1 min,
then increased by 4 °C min-1
to 240 °C, and held for 16 min
at the final temperature.After the run, peaks corresponding
to each FA species were identified by their characteristic
retention times. Concentrations of each sample were nor-
malized against the internal control.
Analysis of gene expression by RT-PCR and
RT-quantitative PCR
Flowers were tagged with different coloured threads to
indicate the days after pollination. Only the seeds from the
siliques on primary shoots were harvested for RNA extrac-
tion. All the RNA samples utilized for verifying the trans-
genic plants were isolated from the young leaves at the
rosette stage. The RNA samples were isolated using the
Invisorb Spin Plant RNA Mini Kit (Invitek, Berlin,
Germany) following the manufacturer’s instructions. The
procedures of semiquantitative RT-PCR (sRT-PCR) and
RT-quantitative PCR (RT-qPCR) were according to our
previous descriptions (Pak et al. 2009).All primer pairs used
in the RT-PCR and RT-qPCR analysis are listed in Support-
ing Information Table S2.
Analysis of seed germination rate and
seedling establishment
Arabidopsis seeds placed on MS medium containing 6%
(w/v) glucose were kept at 4 °C for 3 d to synchronize ger-
mination. The seed germination frequency (defined as
radicle emergence) was scored daily. Arabidopsis plants
(3.5 weeks old) on the MS medium were carefully trans-
ferred into MS solution containing 15% (w/v) polyethylene
glycol 6000 (PEG6000), dehydrated, and harvested 20 min
and 1 h after the PEG6000 treatment for RNA analysis.
Statistical analysis
Completely randomized block design with at least three
biological replicates for each experiment was applied in the
present study.The baseline and threshold cycles (CT value)
were automatically determined using Bio-Rad iQ Software
(version 3.0, Hercules, CA, USA). The relative amounts of
expressed RNA were calculated using the method by Livak
& Schmittgen (2001). Data were classified with Win-Excel
and analysed via analysis of variance (ANOVA) using the
SPSS (version 8.0, SPSS Inc., Chicago, IL, USA) statistical
package. Comparisons between the treatment means were
made using Tukey’s test at a probability level of P Յ 0.05.
RESULTS
Loss of DELLA function changes seed
morphology and FA storage
To determine whether the loss of DELLA function affects
seed morphology and FA storage, WT plants (Ler) were
GA regulates seed fatty acid storage via SFARs 2157
© 2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 2155–2169

4. compared with a Quadruple/ga1–3 (Q/ga1–3) mutant that
contains the loss-of-function gai-t6, rga-t2, rgl1-1 and rgl2-1
alleles in the GA-deficient background (ga1–3). To exclude
effects of GA deficiency, the WT (Ler) was also compared
with a Q/WT mutant that contains the loss-of-function gai-
t6, rga-t2, rgl1-1 and rgl2-1 alleles in the WT (Ler) back-
ground. As shown in Fig. 1, the loss of DELLA function
caused obvious changes in seed morphology. Both the
Figure 1. Comparison of seed morphology among the WT (Ler), the Q/ga1–3 mutants (ga1–3, gai-t6, rga-t2, rgl1-1 and rgl2-1) of the Ler
background, the Q/WT mutants (gai-t6, rga-t2, rgl1-1 and rgl2-1) of the Ler background, the Ler plants treated with GA3 (500 mm), the WT
(Col-0) and the Col-0 plants treated with GA3 (500 mm). Bar = 500 mm. GA3, gibberellin A3; Ler, Landsberg erecta; WT, wild type.
2158 M. Chen et al.
© 2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 2155–2169

5. Q/ga1–3 and the Q/WT plants had greener seeds on the
16th day after pollination (DAP), shorter siliques and less
numerous seeds of a silique upon maturation (data not
shown). Both mutants had lower total FAs per milligram
of seeds. Approximately 1 mg of seeds of Q/ga1–3 and
Q/WT contained only 246.11 and 242.18 mg, respectively, of
total FAs, which was significantly lower compared with
303.38 mg of the WT seeds (Table 1). Notably, the loss of
DELLA function changed the FA composition. Compared
with the WT (Ler), the DELLA mutants had FA compo-
sitions with increased C18:1 and C20:1, and decreased
C18:2 and C18:3 (Table 2). Aside from these changes, the
loss of DELLA function resulted in significant increases in
plant height, silique number and a significant reduction in
branch number (Table 1).
GA triggers DELLA degradation to decrease DELLA
function. We sprayed exogenous GA3 (500 mm) on the WT
plants. Both the Ler and Col-0 plants that were treated with
exogenous GA3 had shorter siliques and fewer seeds per
silique (data not shown). The average weight per thousand
seeds of the treated Col-0 plants was 33.10 mg, 62.2% more
than that of the untreated plants. The weight per thousand
seeds of the treated Ler was 6.89% more than that of the
untreated control. The GA3-treated plants of both the Ler
and Col-0 ecotypes had significantly decreased total seed
FA (mg per mg) featured with enlarged proportions of
C18:1 and C20:1, a decreased proportion of C18:2 and
C18:3 (Tables 1 & 2; Supporting Information Table S4-1,2).
However, the changes were moderate, relative to that of the
DELLA mutants (Table 2).
Overall, the loss of DELLA function through DELLA
mutation or exogenous GA3 treatment reduces the total
seed FA storage, changes the seed FA composition and
causes a variety of morphologic changes.
Q/ga1–3 oil bodies are smaller than those of
Ler upon maturation
The structure of seed oil bodies were examined to investi-
gate the subcellular structural features that lower the FA
content of DELLA mutant seeds. The oil body sizes of Ler
and Q/ga1–3 mutants were compared. At 12 DAP, the
Q/ga1–3 seeds had larger developing oil bodies than the Ler
seeds (Fig. 2). In the mature seeds, however, the Q/ga1–3 oil
bodies were 0.68 (Ϯ0.03) mm along the long axis and 0.48
(Ϯ0.04) mm along the short axis on average, much smaller
Table 1. Comparison for plant morphological traits and seed FA storage among WT (Col-0 and Ler), the WT plants treated with 500 mm
GA3, and the Q/ga1–3, Q/WT mutants
Traits Col-0 Col-0 + GA3 Ler Ler + GA3 Q/ga1–3 Q/WT
Plant height (cm) 24.75 Ϯ 1.23 37.60 Ϯ 4.63* 17.50 Ϯ 2.18 28.95 Ϯ 2.89* 29.76 Ϯ 3.05* 30.03 Ϯ 2.98*
Silique number 23.38 Ϯ 1.51 31.25 Ϯ 4.46* 21.52 Ϯ 3.25 39.78 Ϯ 3.56* 43.28 Ϯ 4.62* 43.98 Ϯ 4.35*
Branch number 4.63 Ϯ 0.52 4.63 Ϯ 0.92 4.68 Ϯ 0.57 4.78 Ϯ 0.48 1.51 Ϯ 0.83* 1.55 Ϯ 0.78*
1000 seed weight (mg) 19.92 Ϯ 1.74 33.10 Ϯ 2.28* 18.58 Ϯ 1.63 19.86 Ϯ 1.58 19.36 Ϯ 1.86 19.52 Ϯ 1.79
Total FAs (mg per mg) 297.36 Ϯ 4.62 253.22 Ϯ 8.09* 303.68 Ϯ 7.08 248.32 Ϯ 4.87* 246.11 Ϯ 4.56* 242.18 Ϯ 4.96*
Total FAs (mg per seed) 5.92 Ϯ 0.38 8.38 Ϯ 0.49* 5.64 Ϯ 0.35 4.93 Ϯ 0.33* 4.76 Ϯ 0.32* 4.73 Ϯ 0.38*
Oil content (%) 29.74 Ϯ 1.98 25.32 Ϯ 1.69* 30.37 Ϯ 1.98 24.83 Ϯ 1.86* 24.61 Ϯ 1.69* 24.22 Ϯ 1.59*
Asterisk (*) indicates the significant difference (P Յ 0.05) compared with the respective control.
FA, fatty acid; GA3, gibberellin A3; Ler, Landsberg erecta; WT, wild type.
Table 2. Reduction between the values of individual FA species of the SFAR gain-of-function plants and those of their corresponding
controls (mol %)
16:0 18:0 18:1 18:2 18:3 20:0 20:1 22:0 22:1 24:0
Col-0 + GA3 -0.79 -0.18 +2.51 -2.99 -0.18 -0.11 +1.87 -0.03 +0.04 -0.03
Ler + GA3 +0.28 +0.66 +6.84 -2.87 -7.44 -0.08 +2.42 +0.06 -0.20 -0.01
Q/ga1–3 +0.52 +0.61 +7.14 -3.02 -7.17 -0.08 +2.33 +0.16 -0.22 0.01
Q/WT +0.20 +0.62 +8.58 -3.49 -7.52 -0.11 +1.34 -0.09 -0.32 -0.02
35S::SFAR1 +0.14 -0.12 +0.54 -1.41 -0.22 -0.10 +1.32 -0.12 -0.17 -0.09
35S::SFAR2 -0.16 -0.17 +1.39 -1.38 -0.64 -0.14 +1.23 -0.11 -0.11 -0.07
35S::SFAR3 -0.18 -0.09 +0.47 -1.18 -0.31 -0.09 +1.48 -0.10 -0.19 -0.02
35S::SFAR4 -0.64 -0.11 +1.59 -0.68 -1.27 -0.18 +1.13 -0.11 -0.18 -0.08
35S::SFAR5 +0.41 -0.17 +0.29 -2.31 -2.89 -0.11 +1.03 -0.10 -0.16 -0.01
A genotype/treatment with a solid star ( ) has Ler as the control, whereas a genotype/treatment with a hollow star ( ) has Col-0 as the
control.The deduction is based on the data in Supporting Information Tables S1-2 and S2-2, where each value is an average of three biological
repetitions. Data for each overexpressed SFAR are based on an average of five to six independent transgenic lines.The bold font indicates the
SFAR feature of FA composition.
FA, fatty acid; Ler, Landsberg erecta; SFAR, Seed Fatty Acid Reducer; WT, wild type.
GA regulates seed fatty acid storage via SFARs 2159
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6. than the 0.95 (Ϯ0.11) mm ¥ 0.62 (Ϯ0.06) mm Ler oil bodies
(Fig. 2; Supporting Information Table S5-1).
Relief of DELLA-dependent repression
up-regulated the TFs that regulate seed
development and genes that catalyze various
steps of FA biosynthesis
The expression of the key TFs (WRI1, FUS3, LEC1, LEC2
and ABI3), which are important for embryogenesis and
seed development in Arabidopsis, was studied by perform-
ing quantitative RT-PCR using the RNAs extracted from
seeds at 6, 10, 12, 14 and 16 DAP (upper panel of Fig. 3). In
WT (Ler), the expression of LEC1 and LEC2 peaked early
at 6 DAP stage. In contrast, the expression of FUS3 and
ABI3 peaked late at 14 and 16 DAP, respectively. The
expression of WRI1 peaked at 10 DAP, later than that of
LEC1 and LEC2, but earlier than FUS3 and ABI3. Relative
to the WT, the Q/ga1–3 mutation caused more than twofold
higher LEC1 and LEC2 transcripts at 10 DAP. It resulted in
1.7-fold higher FUS3 transcripts and nearly threefold
higher WRI1 transcripts at 14 and 16 DAP, and 2.5-fold
higher ABI3 transcripts at 16 DAP.
YUC4, YUC2, IAA17, ACS4, oleosin and 2S3 are down-
stream targets of LEC2 (Stone et al. 2008). Resulting from
the up-regulated LEC2 expression, these genes were also
up-regulated during seed development, in particular at 14
DAP (Supporting Information Fig. S2).
The expression of genes that encode the critical FA bio-
synthetic enzymes CAC2, CAC3, BCCP2 (subunits of
ACCase), MOD1, KASII, CDS2, FAB2, FatA, FAD2,
FAD3 and FAE1 were also examined (middle and bottom
panels of Fig. 3). In the WT, the expression of all of these
genes peaked relatively early, either at 6 DAP, such as the
expression of CAC2, CAC3, BCCP2, MOD1, KASII, CDS2
and FatA, or at 10 DAP, such as the expression of FAB2,
FAD2, FAD3 and FAE1. Relative to the WT, the Q/ga1–3
mutation led to higher transcripts of most of these genes at
relatively later stages. It resulted in more than twofold
increase in the expression of CAC2, BCCP2, MOD1, CDS2,
FatA, FAD3 and FAE1 transcripts at 12 and 14 DAP.
Among the genes that encode the critical FA synthesis
enzymes, KASII was the exception; its expression was
slightly down-regulated in the Q/ga1–3 mutant at almost all
time points.
The expression patterns of these TFs and FA biosynthetic
genes following treatment of the Ler plants with exogenous
GA3 (500 mm) mirrored the results obtained from the
Q/ga1–3 mutant, although the increase in transcription
levels relative to the control was greater in the Q/ga1–3
mutant plants than the GA3-treated Ler plants (Supporting
Information Fig. S3).
Figure 2. Subcellular differences in seed cells from the WT (Ler) and the Q/ga1–3 mutant at 12 DAP and mature seed stage. Arrows
indicate lipid droplets (L), aleurone grains (A) and globoids (G). Bar = 1 mm. DAP, days after pollination; Ler, Landsberg erecta; WT, wild
type.
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7. Overall, the loss of DELLA function up-regulated the
expression of TFs and their corresponding downstream
targets, which implies an increase of seed FA synthesis.
Relief of DELLA repression up-regulated five
GDSL-type SFAR genes that function in
reducing seed FA storage
Based on microarray analysis, Cao et al. (2006) reported
that DELLA proteins down-regulate GDSL-type genes in
unopened flowers and/or imbibed seeds. We designated
these genes as Seed Fatty Acid Reducer (SFARs), specifi-
cally, SFAR1 for At1G54790, SFAR2 for At1G58430, SFAR3
for At2G42990, SFAR4 for At3G48460 and SFAR5 for
At4G18970. The SFAR expression in developing Q/ga1–3
seeds was examined at 6, 10, 12, 14 and 16 DAP.Those of the
Q/WT were examined at 12, 14 and 16 DAP. Figure 4 clearly
shows that the SFARs in both the Q/ga1–3 and the Q/WT
were significantly up-regulated at 14 DAP by up to 60-fold
more than in the WT (Ler). Again, the GA3 treatment
(500 mm) produced similar changes in SFAR expression.
However, their peak increases were much lower (Support-
ing Information Fig. S4).
Interruption or overexpression of the SFARs
altered total seed FA content and composition
To clarify further the role of five SFARs in seed FA storage,
the total seed FA content and FA species composition of
WT (Col-0), sfar single and double mutants, the sfar double
mutants sfar2-1 sfar3-1, and sfar4-1 sfar5-1 plants treated
with GA3 (500 mm) and the transgenic lines that overexpress
individual SFARs were compared (Fig. 5; Supporting Infor-
mation Table S6). As shown in Fig. 5, the interruption of an
individual SFAR gene resulted in significant increases in
total seed FA from 9.1 to 16.9%, depending on the locus
interrupted. In contrast, the ectopic SFAR overexpression
resulted in significant reductions in total seed FA from 13.4
to 25.1%, depending on the overexpressed gene. The over-
expression of any of the five SFARs resulted in the change
of FA composition, featured with an increase of C18:1 and
C20:1, and a decrease of C18:2 and C18:3 proportions
(Table 2 & Supporting Information Table S6).
Double mutations of the SFARs were generated to deter-
mine if the SFAR loci had additive effects on seed FA
storage. Except for the double mutant sfar1 sfar2 that was
difficult to produce because of the low recombination rate,
nine double mutants were generated and harvested for seed
FA analysis. On average, the double mutants showed higher
increases in total seed FA (23.7%) compared with the single
mutants (13.4%). The double mutant sfar1-3 sfar3-1 exhib-
ited a 31.8% increase in total seed FA compared with the
WT (Fig. 5; Supporting Information Table S6-1).
The seed morphology and oil body structure of the WT
(Col-0), the SFAR gain-of-function transgenic plants
(35S::SFAR1), and the loss-of-function mutants (sfar2-1
sfar3-2) were further compared. As shown in Fig. 6, the
seed morphology of the 35::SFAR1 plants and sfar2-1
Figure 3. Comparison of the relative transcription levels of the TFs that regulate embryogenesis and seed development (top panel), and
the genes that encode enzymes critical to FA biosynthesis (middle and bottom panels) between the WT (Ler) and the Q/ga1–3 mutants at
different seed developmental stages. The transcription levels are relative to the WT (Ler) at 6 DAP, which is set to 1. FA, fatty acid; DAP,
days after pollination; Ler, Landsberg erecta; TF, transcription factor; WT, wild type.
GA regulates seed fatty acid storage via SFARs 2161
© 2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 2155–2169

8. sfar3-2 plants were not visibly different from that of
the WT (Col-0). However, significant differences were
observed among the WT, 35S::SFAR1 and sfar2-1 sfar3-2
plants in terms of average number and size of oil bodies in
the seed cells. A sfar2-1 sfar3-2 seed cell contained an
average of 171.06 (Ϯ14.72) visible oil bodies, less than
the 386.66 (Ϯ19.52) oil bodies in WT seed cells and the
453.91 (Ϯ12.72) oil bodies in 35S::SFAR1 seed cells. On the
other hand, the sfar2-1 sfar3-2 oil bodies measured 1.69
(Ϯ0.23) mm along the long axis and 0.87 (Ϯ0.12) mm along
the short axis, much larger than the 0.69 (Ϯ0.08) mm ¥ 0.61
(Ϯ0.05) mm WT oil bodies and the 0.48 (Ϯ0.03) mm ¥ 0.31
(Ϯ0.02) mm 35S::SFAR1 oil bodies (Supporting Informa-
tion Table S5-2).
Therefore, gain of SFAR function reduces seed FA
storage and oil body size, whereas loss of SFAR function
increases seed FA storage and oil body size. Furthermore,
the SFAR loci have an additive effect on seed FA content.
SFAR gene overexpression enhanced the
germination rate and young seedling
establishment in a stressed environment
Considering FA metabolism may be involved in stress-
resistance mechanisms, and SFAR overexpression changes
the seed FA composition in the transgenic plants, we inves-
tigated whether SFAR gene overexpression enhances the
germination rate and seedling establishment under stressed
environments. On medium containing 6% (w/v) glucose,
60% of the seeds from the 35S::SFAR4 transgenic line #12
and 50% of the seeds from the 35S::SFAR4 transgenic line
#23 germinated on the third day after sowing, whereas only
3% of the WT seeds germinated. Seven days after sowing,
only 35% of the WT seed germinated, whereas the germi-
nation rates of transgenic lines #12 and #23 were 98 and
97%, respectively (Fig. 7a). In 6% (w/v) glucose, about 55 to
65% of the transgenic plants reached the two-leaf stage 3.5
weeks after sowing. By contrast, the WT plants failed to
develop normal cotyledons and green leaves under the
same stressful condition (Fig. 7b). The overexpression of
the other four Arabidopsis SFAR genes, viz., SFAR1,
SFAR2, SFAR3 and SFAR5, had similar effects on glucose
tolerance during seed germination (Table 3). We also com-
pared between theWT and sfar loss-of-function mutants for
tolerance against osmotic stress; and we didn’t find any
significant differences in germination rate and young seed-
ling establishment (data not shown).
The transient expressional differences between genes
related to osmotic stress in the WT (Col-0) and the
35S::SFAR4 plants were compared using sRT-PCR
(Fig. 7c).The osmotic stress caused by short-term PEG6000
treatment induced the differential expression of genes such
as ABA1, ABA2, ABA3, ABI1, ADH, Rab18, CBL1, CBL9
and NCED3 between the WT and the transgenic plants.
After 60 min of PEG6000 treatment, the genes had higher
expression levels in the WT (Col-0) than in the transgenic
Figure 4. Comparison of the relative transcription levels of SFARs between the WT (Ler) and the Q/ga1–3 mutant at different seed
developmental stages. The transcription levels of the WT at 6 DAP are set to 1 for the upper panel. The transcription levels of the WT at
12 DAP are set to 1 for bottom panel. DAP, days after pollination; Ler, Landsberg erecta; SFAR, Seed Fatty Acid Reducer; WT, wild type.
2162 M. Chen et al.
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9. plants, which indicates that the transgenic plants have
reduced sensitivity to osmotic stress.
DISCUSSION
Previous studies reported that the GA signal regulates
plant growth and development, such as seed germination,
stem elongation and leaf expansion, as well as trichome,
flower and fruit development (Swain et al. 1997; King et al.
2001; Sasaki et al. 2003; Cheng et al. 2004; Sun et al. 2004;
Fleet & Sun 2005; Yamauchi et al. 2007). However, little
attention has been paid on the effects of GA signalling on
the regulation of FA storage in seeds and the downstream
factors that positively or negatively determine seed FA
storage capacity. In the present study, the knockout of
DELLA proteins or the application of GA3 to WT plants
significantly reduced seed FA storage in Arabidopsis. This
effect on seed FA requires the action of SFAR because
the FA composition in the DELLA loss-of-function plants
clearly featured an ‘SFAR footprint,’ that is, increased
C18:1 and C20:1, and decreased C18:2 and C18:3, accom-
panied by a reduction in the total seed FA content
(Table 2). On the other hand, the GA3-treated sfar2-1
sfar3-2 and/or sfar4-2 sfar5-1 double mutant plants had a
lower FA content than the untreated double mutant
plants. They had the ‘SFAR footprint’ nevertheless, indi-
cating that GA would have up-regulated unmutated
SFARs, greater in number than the genes mutated. The
up-regulation of these SFARs offset the increase of seed
FA in the double mutants that would otherwise be
observed (Fig. 5). The best way to demonstrate the SFAR
requirement of GA is to treat SFAR-silenced plants with
GA3 and the resulting seed FA content. However, this
treatment is technically difficult to perform. Firstly, the
generation of sfar1 sfar2 is difficult because of the close
linkage of the two genes on one chromosome. Secondly,
the exact number of SFARs acting on the GA signalling
pathway is still unknown. GA may affect the seed FA
content via SFARs (with the ‘SFAR footprint’). However,
GA may also affect the seed FA content independent of
SFARs. As shown in Table 1, the GA signal affects many
plant traits. Traits such as plant height, as well as silique
and branch numbers affect the seed oil content, a typical
quantitative trait regulated by many factors. Moreover,
GA functions via the DELLA-dependent pathway as
well as independently in Arabidopsis (Cao et al. 2005).
Nonetheless, the GA-SFAR pathway is an important
GA-mediated regulator of seed FA content.
Figure 5. Comparison of the seed FA content between the WT (Col-0) and the SFAR single gene mutants, the transgenic plants that
overexpress the respective single SFAR genes (a–e) and the double mutants (f). The dashed lines indicate the level of seed FA content in
WT (Col-0). The asterisks (*) indicate significant difference (P Յ 0.05) compared with the WT. FA, fatty acid; SFAR, Seed Fatty Acid
Reducer; WT, wild type.
GA regulates seed fatty acid storage via SFARs 2163
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10. The loss of DELLA function up-regulated the TFs, such
as LEC1, LEC2, FUS3, ABI3 and WRI1, which regulate
embryonic and seed development (upper panel of Fig. 3;
Supporting Information Fig. S3). In Arabidopsis, LEC1 is a
key regulator of FA biosynthesis, and LEC1 overexpression
increased the expression of many FA biosynthetic genes
involved in the condensation, chain elongation and desatu-
ration of FA biosynthesis (Mu et al. 2008). The LEC2
protein induces the maturation of many traits, auxin activ-
ity, and the expression of the other seed regulators like
FUS3 and ABI3, which redundantly induce the expression
of seed storage proteins, degrading chlorophyll and
Figure 6. Comparison of the seed morphology and oil body structure among the WT (Col-0), the gain-of-function line that
overexpresses SFAR1 and the loss-of-function line sfar2-1 sfar3-2. Arrows indicate lipid droplets (L), aleurone grains (A) and globoids
(G). Bar = 500 mm for the left panels; bar = 1 mm for the right panels. SFAR, Seed Fatty Acid Reducer; WT, wild type.
2164 M. Chen et al.
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11. anthocyanins in dry seeds, and confers sensitivity to ABA
(Luerssen et al. 1998; To et al. 2006). Moreover, LEC2 regu-
lates seed FA metabolism by targeting WRI1, which con-
verts sucrose into precursors of TAG biosynthesis (Cernac
& Benning 2004; Baud et al. 2007) by targeting oleosin,
which maintains oil bodies as small single units and pre-
vents their coalescence during seed desiccation (Siloto et al.
2006). The sizes of the oil bodies in the plants with reduced
DELLA function and/or increased SFAR expression were
smaller than that in WT (Figs 2 & 6). This may arise from
two reasons. Firstly, LEC2 and its target gene oleosin are
up-regulated in these plants. Secondly, the SFARs, which
may involve FA hydrolyzation before free FAs are incor-
porated into lipid droplets, are also up-regulated in these
plants. However, Siloto et al. (2006) also reported that the
interruption of oleosin with RNAi produces unusually large
oil bodies that correlate with lower seed lipid content. In
contrast to their results, the current observations indicate
that larger oil bodies correlate with higher final seed FA
content. LEC2 is also involved in the auxin response (Stone
Figure. 7. Comparison of the germination rate (a) and establishment of young seedlings (b) between the WT (Col-0) and the transgenic
lines (#12, #23) that overexpress SFAR4 on MS medium containing 6% (w/v) glucose. (c) RT-PCR results comparing the gene expression
induced by osmotic stress between the WT (Col-0) and the transgenic line #12 that overexpresses SFAR4. The expression levels of the
genes were compared at 0, 20 and 60 min after PEG6000 treatment. The differentially expressed genes are highlighted in the white line
boxes. MS, Murashige and Skoog; PEG6000, polyethylene glycol 6000; WT, wild type.
Table 3. Comparison of germination rate
(%) on MS medium containing 6% (w/v)
glucose between the WT (Col-0) and the
transgenic lines that overexpress SFAR1,
SFAR2, SFAR3 and SFAR5
1 DAS 2 DAS 3 DAS 4 DAS 5 DAS 6 DAS 7 DAS
WT(Col-0) 0 Ϯ 0 0 Ϯ 0 3 Ϯ 1 5 Ϯ 2 15 Ϯ 2 23 Ϯ 3 35 Ϯ 4
35S::SFAR1 #6 0 Ϯ 0 0 Ϯ 0 45 Ϯ 3 95 Ϯ 4 97 Ϯ 3 98 Ϯ 2 98 Ϯ 2
35S::SFAR1 #15 0 Ϯ 0 0 Ϯ 0 50 Ϯ 3 87 Ϯ 5 95 Ϯ 3 95 Ϯ 4 98 Ϯ 2
35S::SFAR2 #1 0 Ϯ 0 0 Ϯ 0 35 Ϯ 2 80 Ϯ 5 90 Ϯ 5 95 Ϯ 3 97 Ϯ 3
35S::SFAR2 #8 0 Ϯ 0 0 Ϯ 0 40 Ϯ 3 75 Ϯ 5 90 Ϯ 5 93 Ϯ 4 97 Ϯ 2
35S::SFAR3 #2 0 Ϯ 0 0 Ϯ 0 55 Ϯ 3 85 Ϯ 4 93 Ϯ 4 97 Ϯ 3 98 Ϯ 1
35S::SFAR3 #8 0 Ϯ 0 0 Ϯ 0 50 Ϯ 3 78 Ϯ 4 90 Ϯ 5 95 Ϯ 4 98 Ϯ 2
35S::SFAR5 #3 0 Ϯ 0 0 Ϯ 0 50 Ϯ 2 85 Ϯ 4 90 Ϯ 3 95 Ϯ 3 98 Ϯ 2
35S::SFAR5 #5 0 Ϯ 0 0 Ϯ 0 55 Ϯ 2 80 Ϯ 3 86 Ϯ 5 97 Ϯ 2 98 Ϯ 1
DAS is days after sowing.
# is the individual transgenic line number.
The germination rates of two 35S::SFAR4 transgenic lines are presented in Fig. 7a.
SFAR, Seed Fatty Acid Reducer; WT, wild type.
GA regulates seed fatty acid storage via SFARs 2165
© 2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 2155–2169

12. et al. 2008).Therefore, fewer branches were observed in the
DELLA mutant plants, possibly due to enhanced apical
dominance (Table 1). The GA3 treatment did not reduce
branching in the WT plants because the branches and
lateral buds should have already formed before the appli-
cation of GA3 (Table 1).
The up-regulation ofTFs during seed development by the
loss of DELLA function resulted in increased transcription
of genes that catalyze various steps in the formation of
different FA species, which include CAC2, CAC3, BCCP2,
MOD1, FAB2, CDS2, FAD2, FAD3 and FAE1 (middle and
bottom panels of Fig. 3; Supporting Information Fig. S3). Of
these enzymes, CAD2, CAC2 and BCCP2 are subunits of
ACCase, which catalyze the initial synthesis step, the for-
mation of malonyl-CoA from acetyl-CoA.The MOD1 func-
tions as an enoyl-ACP reductase. FAB2 (SSI2) encodes a
stearoyl-ACP desaturase (Lightner et al. 1994). The CDS2
enzyme determines the phosphatidate cytidylyl transferase
activity and is involved in the phospholipid biosynthetic
process. FAD2 is essential for polyunsaturated lipid synthe-
sis (Okuley et al. 1994) and FAD3 is responsible for the
synthesis of 18:3 FAs from phospholipids (Shah, Xin &
Browse 1997). FAE1 is required for the synthesis of very
long chain FAs in seeds and it is presumed to be a condens-
ing enzyme that extends the chain length of FAs from C18
to C20 and C22 (James et al. 1995). Both SFAR gain-of-
function plants and DELLA loss-of-function plants had a
significantly increased proportion of C18:1 and C20:1. The
up-regulated expressions of the genes such as FAB2 and
FAE1 could be the probable reason responsible for the
feature (Table 2; Fig. 3; Supporting Information Fig. S3).
The up-regulation of the TFs and the downstream FA
biosynthesis genes induced by the relief of DELLA repres-
sion implies higher seed FA storage. However, the total
seed FA decreased significantly (Table 1; Supporting Infor-
mation Table S4). This may be because the loss of DELLA
function also accelerates the seed FA breakdown by
up-regulating SFARs.The formation of seed FA should be a
dynamic balance between synthesis and breakdown. SFARs
were up-regulated in the Q/ga1–3 plants from 12 DAP and
peaked at 14 DAP (Fig. 4). Previous studies have shown
that embryo morphogenesis is accomplished at 6 DAP, and
the accumulation of storage compound starts from 7 DAP
and is completed at 20 DAP, which is essential for seed FA
accumulation from 12 DAP to 16 DAP (Goldberg et al.
1994; Baud et al. 2002; Fait et al. 2006; Baud & Lepiniec
2009). Most of the aforementioned FA synthesis genes were
up-regulated at 12 DAP or at earlier stages, whereas the
SFAR genes were drastically up-regulated at 14 DAP. In
line with this observation,the Q/ga1–3 mutant had larger oil
bodies than the WT at 12 DAP, but smaller oil bodies at the
mature stage after the drastic SFAR expressional peak at 14
DAP (Figs 2 & 4). Taken together, FA synthesis and break-
down, the opposing processes that determine the final
quantity of seed FA storage, are subjected to global regu-
lation by TFs during seed development.
Our results demonstrate that SFARs, the GDSL-motif
genes, involve seed FA breakdown and lead to a less seed
FA storage (Fig. 5). SFARs might cause less free FAs to be
incorporated into lipid droplets, where TAG is the main
constituent. To the best of our knowledge, there has not
been any evidence demonstrating that a plant GDSL-motif
gene has any kind of lipase activity. Our experiment indi-
rectly confirmed that SFARs are not lipases. If they were,
higher free FA contents in seeds of those SFAR gain-of-
function genotypes would have been detected. In fact,
SFARs cause less seed FA storage. We show that somehow
SFARs involve the seed FA breakdown. However, the exact
role of SFAR remains unclear and need further investiga-
tion. So far, the b, a and w-oxidation pathways are under-
stood for FA decomposition and b-oxidation pathway is the
general model of FA catabolism in planta (Harwood 1988;
Graham & Eastmond 2002).
In addition to the change in seed FAs, the loss of DELLA
function also produced enlarged seeds, with a fewer number
of seed sets and shorter siliques. More photosynthates
would have been transported to these larger seeds. Inter-
estingly, the application of GA3 did not alter the shape of
the Col-0 ecotype seeds, but altered those of the Ler
ecotype. Moreover, both the Q/ga1–3 and Q/WT mutation
changed the seed shape from oval to round (Fig. 1). Differ-
ences in the seed formation were observed between the two
ecotypes in response to the loss of DELLA function.
Interestingly,the transgenic line that overexpresses one of
the SFAR esterase genes was much less sensitive to stressful
environments,as reflected by their much higher germination
rate and better young seedling establishment than the WT
(Col-0) (Fig. 7a,b). On the other hand, the loss-of-function
sfar mutants were not significantly different from the WT
plants in terms of stress tolerance. Higher stress tolerance
may result from the ‘SFAR footprint’, that is, a change in FA
composition characterized by increased C18:1 and C20:1,
and decreased C18:2 and C18:3. The ‘SFAR footprint’
was only found in SFAR gain-of-function plants, whereas
no significant differences in seed FA composition were
observed between the sfar mutants and theWT plants (Sup-
porting InformationTable S6-1).FAs are major components
of the phospholipid bilayer and are involved in several cell
membrane functions. FA metabolism is involved in the
stress-resistance mechanisms of some species (Horikawa &
Sakamoto 2009).A decrease in polyunsaturated FAs report-
edly reduces the osmotic stress tolerance of transgenic
worms (Horikawa & Sakamoto 2009;Tazearslan et al.2009).
However, the role of FA unsaturation in stress tolerance
and the interaction between FA metabolism and stress
responses in plants remain poorly understood.
In Arabidopsis, there are 108 GDSL-type genes (Ling
2008).The five SFARs that were characterized in this paper
are regulated by GA signalling.The regulation of the rest of
the GDSL-type genes, and whether they modulate seed oil
formation remain interesting questions.
ACKNOWLEDGMENTS
We thank Prof Dr Xiangdong Fu for providing the Q/WT
mutant used in the study.The work of our lab was supported
2166 M. Chen et al.
© 2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 2155–2169

13. by the Natural Science Foundation of China (Grant nos.
30971700 and 31171463) and Zhejiang Province (Grant no.
Z3100130), the Fundamental Research Funds for the
Central Universities (2012FZA6011 and 2012XZZX012),
and the Special Grand National Science and Technology
Project (Grant no. 2009ZX08009-076B).
REFERENCES
Akoh C.C., Lee G.C., Liaw Y.C., Huang T.H. & Shaw J.F. (2004)
GDSL family of serine esterases/lipases. Progress in Lipid
Research 43, 534–552.
Baud S. & Lepiniec L. (2009) Regulation of de novo fatty acid
synthesis in maturing oilseeds of Arabidopsis. Plant Physiology
and Biochemistry 47, 448–455.
Baud S., Boutin J.P., Miquel M., Lepiniec L. & Rochat C. (2002) An
integrated overview of seed development in Arabidopsis
thaliana ecotypeWS.Plant Physiology and Biochemistry 40, 151–
160.
Baud S., Mendoza M.S., To A., Harscoet E., Lepiniec L. &
Dubreucq B. (2007) WRINKLED1 specifies the regulatory
action of LEAFY COTYLEDON2 towards fatty acid metabo-
lism during seed maturation in Arabidopsis. The Plant Journal
50, 825–838.
Baud S., Wuilleme S., To A., Rochat C. & Lepiniec L. (2009) Role
of WRINKLED1 in the transcriptional regulation of glycolytic
and fatty acid biosynthetic genes in Arabidopsis. The Plant
Journal 60, 933–947.
Beisson F., Gardies A.M., Teissere M., Ferte N. & Noat G. (1997)
An esterase neosynthesized in post-germinated sunflower seeds
is related to a new family of lipolytic enzymes. Plant Physiology
and Biochemistry 35, 761–765.
Cao D.N., Hussain A., Cheng H. & Peng J.R. (2005) Loss of
function of four DELLA genes leads to light- and gibberellin-
independent seed germination in Arabidopsis. Planta 223, 105–
113.
Cao D.N., Cheng H., Wu W., Soo H.M. & Peng J.R. (2006) Gibber-
ellin mobilizes distinct DELLA-dependent transcriptomes to
regulate seed germination and floral development in Arabidop-
sis. Plant Physiology 142, 509–525.
Cernac A. & Benning C. (2004) WRINKLED1 encodes an AP2/
EREB domain protein involved in the control of storage
compound biosynthesis in Arabidopsis. The Plant Journal 40,
575–585.
Cheng H., Qin L.J., Lee S.C., Fu X.D., Richards D.E., Cao D.N., Luo
D., Harberd N.P. & Peng J.R. (2004) Gibberellin regulates Ara-
bidopsis floral development via suppression of DELLA protein
function. Development 131, 1055–1064.
Clough S.J. & Bent A.F. (1998) Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis
thaliana. The Plant Journal 16, 735–743.
Dill A. & Sun T.P. (2001) Synergistic derepression of gibberellin
signaling by removing RGA and GAI function in Arabidopsis
thaliana. Genetics 159, 777–785.
Eastmond P.J. (2006) SUGAR-DEPENDENT1 encodes a patatin
domain triacylglycerol lipase that initiates storage oil breakdown
in germinating Arabidopsis seeds. The Plant Cell 18, 665–675.
Fait A., Angelovici R., Less H., Ohad I., Urbanczyk-Wochniak E.,
Fernie A.R. & Galili G. (2006) Arabidopsis seed development
and germination is associated with temporally distinct metabolic
switches. Plant Physiology 142, 839–854.
Fleet C.M. & Sun T.P. (2005) A DELLAcate balance: the role of
gibberellin in plant morphogenesis. Current Opinion in Plant
Biology 8, 77–85.
Focks N. & Benning C. (1998) wrinkled1: a novel, low-seed-oil
mutant of Arabidopsis with a deficiency in the seed-specific regu-
lation of carbohydrate metabolism. Plant Physiology 118,
91–101.
Goldberg R.B., Depaiva G. & Yadegari R. (1994) Plant embryo-
genesis – zygote to seed. Science 266, 605–614.
Graham I.A. & Eastmond P.J. (2002) Pathways of straight and
branched chain fatty acid catabolism in higher plants. Progress in
Lipid Research 41, 156–181.
Grindstaff K.K., Fielding L.A. & Brodl M.R. (1996) Effect of gib-
berellin and heat shock on the lipid composition of endoplasmic
reticulum in barley aleurone layers. Plant Physiology 110, 571–
581.
Harwood J.L. (1988) Fatty-acid metabolism. Annual Review of
Plant Physiology 39, 101–138.
Harwood J.L. (1996) Recent advances in the biosynthesis of plant
fatty acids. Biochimica Et Biophysica Acta-Lipids and Lipid
Metabolism 1301, 7–56.
Hays D.B., Yeung E.C. & Pharis R.P. (2002) The role of gibberellins
in embryo axis development. Journal of Experimental Botany 53,
1747–1751.
Horikawa M. & Sakamoto K. (2009) Fatty-acid metabolism is
involved in stress-resistance mechanisms of Caenorhabditis
elegans. Biochemical and Biophysical Research Communications
390, 1402–1407.
James D.W., Jr, Lim E., Keller J., Plooy I., Ralston E. & Dooner
H.K. (1995) Directed tagging of the Arabidopsis FATTY ACID
ELONGATION1 (FAE1) gene with the maize transposon acti-
vator. The Plant Cell 7, 309–319.
Jiang C. & Fu X. (2007) GA action: turning on de-DELLA repress-
ing signaling. Current Opinion in Plant Biology 10, 461–465.
Kagaya Y., Toyoshima R., Okuda R., Usui H., Yamamoto A. &
Hattori T. (2005) LEAFY COTYLEDON1 controls seed
storage protein genes through its regulation of FUSCA3 and
ABSCISIC ACID INSENSITIVE3. Plant and Cell Physiology
46, 399–406.
King K.E., Moritz T. & Harberd N.P. (2001) Gibberellins are not
required for normal stem growth in Arabidopsis thaliana in the
absence of GAI and RGA. Genetics 159, 767–776.
Kroj T., Savino G., Valon C., Giraudat J. & Parcy F. (2003) Regu-
lation of storage protein gene expression in Arabidopsis. Devel-
opment 130, 6065–6073.
Kwon S.J., Jin H.C., Lee S., Nam M.H., Chung J.H., Kwon S.I., Ryu
C.M. & Park O.K. (2009) GDSL lipase-like 1 regulates systemic
resistance associated with ethylene signaling in Arabidopsis. The
Plant Journal: For Cell and Molecular Biology 58, 235–245.
Lee D.S., Kim B.K., Kwon S.J., Jin H.C. & Park O.K. (2009) Ara-
bidopsis GDSL lipase 2 plays a role in pathogen defense via
negative regulation of auxin signaling. Biochemical and Bio-
physical Research Communications 379, 1038–1042.
Lee S., Cheng H., King K.E., Wang W., He Y., Hussain A., Lo J.,
Harberd N.P. & Peng J. (2002) Gibberellin regulates Arabidopsis
seed germination via RGL2, a GAI/RGA-like gene whose
expression is up-regulated following imbibition. Genes and
Development 16, 646–658.
Lightner J., James D.W., Dooner H.K. & Browse J. (1994) Altered
body morphology is caused by increased stearate levels in a
mutant of Arabidopsis. The Plant Journal 6, 401–412.
Ling H. (2008) Sequence analysis of GDSL lipase gene family in
Arabidopsis thaliana. Pakistan Journal of Biological Sciences 11,
763–767.
Livak K.J. & Schmittgen T.D. (2001) Analysis of relative gene
expression data using real-time quantitative PCR and the
2(T)(-Delta Delta C) method. Methods 25, 402–408.
Luerssen K., Kirik V., Herrmann P. & Misera S. (1998) FUSCA3
encodes a protein with a conserved VP1/ABI3-like B3 domain
GA regulates seed fatty acid storage via SFARs 2167
© 2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 2155–2169

14. which is of functional importance for the regulation of seed
maturation in Arabidopsis thaliana. The Plant Journal 15, 755–
764.
Mu J., Tan H., Zheng Q., et al. (2008) LEAFY COTYLEDON1 is a
key regulator of fatty acid biosynthesis in Arabidopsis. Plant
Physiology 148, 1042–1054.
Naranjo M.A., Forment J., Roldan M., Serrano R. & Vicente O.
(2006) Overexpression of Arabidopsis thaliana LTL1, a salt-
induced gene encoding a GDSL-motif lipase, increases salt tol-
erance in yeast and transgenic plants. Plant, Cell & Environment
29, 1890–1900.
O’Neill C.M., Gill S., Hobbs D., Morgan C. & Bancroft I. (2003)
Natural variation for seed oil composition in Arabidopsis
thaliana. Phytochemistry 64, 1077–1090.
Okuley J., Lightner J., Feldmann K., Yadav N., Lark E. & Browse J.
(1994) Arabidopsis Fad2 gene encodes the enzyme that is essen-
tial for polyunsaturated lipid-synthesis.The Plant Cell 6, 147–158.
Pak H.S., Guo Y., Chen M.X., Chen K.M., Li Y.L., Hua S.J., Shamsi
I., Meng H.B., Shi C.G. & Jiang L.X. (2009) The effect of exog-
enous methyl jasmonate on the flowering time, floral organ mor-
phology, and transcript levels of a group of genes implicated in
the development of oilseed rape flowers (Brassica napus L.).
Planta 231, 79–91.
Pan Y.L., Tchagangl A., Berube H., Phan S., Shearer H., Liu Z.Y.,
Fobert P. & Famili F. (2010) Integrative data mining in functional
genomics of Brassica napus and Arabidopsis thaliana. Trends in
Applied Intelligent Systems, Pt Iii, Proceedings 6098, 92–101.
Peng J. & Harberd N.P. (1997) Gibberellin deficiency and response
mutations suppress the stem elongation phenotype of
phytochrome-deficient mutants of Arabidopsis.Plant Physiology
113, 1051–1058.
Sasaki A., Itoh H., Gomi K., et al. (2003) Accumulation of phos-
phorylated repressor for gibberellin signaling in an F-box
mutant. Science 299, 1896–1898.
Shah S., Xin Z.G. & Browse J. (1997) Overexpression of the FAD3
desaturase gene in a mutant of Arabidopsis. Plant Physiology
114, 1533–1539.
Siloto R.M.P., Findlay K., Lopez-Villalobos A., Yeung E.C., Nyki-
foruk C.L. & Moloney M.M. (2006) The accumulation of
oleosins determines the size of seed oilbodies in Arabidopsis.
The Plant Cell 18, 1961–1974.
Silverstone A.L.,Ciampaglio C.N. & Sun T. (1998) The Arabidopsis
RGA gene encodes a transcriptional regulator repressing the
gibberellin signal transduction pathway. The Plant Cell 10, 155–
169.
Silverstone A.L., Jung H.S., Dill A., Kawaide H., Kamiya Y. & Sun
T.P. (2001) Repressing a repressor: gibberellin-induced rapid
reduction of the RGA protein in Arabidopsis. The Plant Cell 13,
1555–1566.
Singh D.P., Filardo F.F., Storey R., Jermakow A.M., Yamaguchi S. &
Swain S.M. (2010) Overexpression of a gibberellin inactivation
gene alters seed development,KNOX gene expression,and plant
development in Arabidopsis. Physiologia Plantarum 138, 74–90.
Stone S.L., Braybrook S.A., Paula S.L., Kwong L.W., Meuser J.,
Pelletier J., Hsieh T.F., Fischer R.L., Goldberg R.B. & Harada J.J.
(2008) Arabidopsis LEAFY COTYLEDON2 induces matura-
tion traits and auxin activity: implications for somatic embryo-
genesis. Proceedings of the National Academy of Sciences of the
United States of America 105, 3151–3156.
Sun M.H., Zhang M.H., Liu H.Y., Li L.G., Yu X., Su W.A. & Tang
Z.C. (2004) Distribution of water channel protein RWC3 and its
regulation by GA and sucrose in rice (Oryza sativa). Acta
Botanica Sinica 46, 1056–1064.
Swain S.M., Reid J.B. & Kamiya Y. (1997) Gibberellins are
required for embryo growth and seed development in pea. The
Plant Journal 12, 1329–1338.
Tazearslan C., Ayyadevara S., Bharill P. & Reis R.J.S. (2009) Posi-
tive feedback between transcriptional, kinase suppression in
nematodes with extraordinary longevity, stress resistance. PLoS
Genetics 5, e1000452.
To A., Valon C., Savino G., Guilleminot J., Devic M., Giraudat J. &
Parcy F. (2006) A network of local and redundant gene regula-
tion governs Arabidopsis seed maturation. The Plant Cell 18,
1642–1651.
Tyler L., Thomas S.G., Hu J.H., Dill A., Alonso J.M., Ecker J.R. &
Sun T.P. (2004) DELLA proteins and gibberellin-regulated seed
germination and floral development in Arabidopsis. Plant Physi-
ology 135, 1008–1019.
Ueguchi-Tanaka M., Nakajima M., Katoh E., et al. (2007) Molecu-
lar interactions of a soluble gibberellin receptor, GID1, with a
rice DELLA protein, SLR1, and gibberellin. The Plant Cell 19,
2140–2155.
Yamauchi Y.,Takeda-Kamiya N.,Hanada A.,Ogawa M.,Kuwahara
A., Seo M., Kamiya Y. & Yamaguchi S. (2007) Contribution of
gibberellin deactivation by AtGA2ox2 to the suppression of ger-
mination of dark-imbibed Arabidopsis thaliana seeds. Plant and
Cell Physiology 48, 555–561.
Yu H., Ito T., Zhao Y.X., Peng J.R., Kumar P.P. & Meyerowitz E.M.
(2004) Floral homeotic genes are targets of gibberellin signaling
in flower development. Proceedings of the National Academy of
Sciences of the United States of America 101, 7827–7832.
Zentella R., Zhang Z.L., Park M., et al. (2007) Global analysis of
DELLA direct targets in early gibberellin signaling in Arabidop-
sis. The Plant Cell 19, 3037–3057.
Received 23 February 2012; received in revised form 14 May 2012;
accepted for publication 18 May 2012
SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article.
Figure S1. Identification of the SALK mutants at the SFAR
loci.
Figure S2. Comparison of the relative transcription levels of
the LEC2 targeting genes between the WT (Ler) and the
Q/ga1-3 mutant.The transcription levels are relative to WT
(Ler) at 6 DAP, which is set to 1.
Figure S3. Comparison of the relative transcription levels of
the TFs that regulate embryogenesis and seed development
and the genes that encode enzymes needed for FA biosyn-
thesis at different seed developmental stages between the
WT (Ler) and GA3-treated WT plants. The transcription
levels are relative to the WT (Ler) at 10 DAP, which is set
to 1.
Figure S4. Comparison of the relative transcription levels of
the five GDSL-type genes at different seed developmental
stages between the WT (Ler) and GA3-treated WT plants.
The transcription levels are relative to the WT (Ler) at 10
DAP, which was set to 1.
Figure S5. Comparison of the germination rates of the WT
(Col-0) and the SFAR transgenic lines.
Table S1. Primers used in genotyping the SALK mutants.
Table S2. Primers used in the RT-PCR and RT-qPCR
analyses.
2168 M. Chen et al.
© 2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 2155–2169

15. Table S3. Primers used for cloning the cDNA of the five
GDSL-type genes.
Table S4. Comparison of the seed FA composition of the
Q/ga1–3, Q/WT, WT (Col-0 and Ler) and the WT plants
treated with 500 mm GA3 (S4-1 in mg per mg and S4-2 in
mol %).
Table S5. Oil body statistics (S5-1 is the comparison
between Ler and the Q/ga1-3 mutant; S5-2 is the
comparison among Col-0, 35S::SFAR1 and sfar2-1
sfar3-2).
Table S6. FA composition of the SFAR mutants and the
transgenic lines (S6-1 in mg mg-1
and S6-2 in mol %).
GA regulates seed fatty acid storage via SFARs 2169
© 2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 2155–2169