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Plant Science 252 (2016) 42–52

Contents lists available at ScienceDirect

Plant  Science

j ourna l ho me pa g e: www.elsev ier .com/ locate /p lantsc i

unflower  HaGPAT9-1  is  the  predominant  GPAT  during  seed
evelopment

iriam  Payá-Milansa,b, Jose  Antonio  Aznar-Morenoa,c, Tiago  S.  Balbuenad,e,
ichard P.  Haslamf,  Satinder  K.  Giddag,  Javier  Pérez-Hormaechea,  Robert  T.  Mulleng,

ay  J. Thelend,  Johnathan  A.  Napier f, Joaquín  J.  Salasa,  Rafael  Garcésa,
nrique  Martínez-Forcea, Mónica  Venegas-Caleróna,∗

Department of Biochemistry and Molecular Biology of Plant Products, Instituto de la Grasa (CSIC), Campus Universitario Pablo de Olavide, 41013 Seville,
pain
Department of Entomology & Plant Pathology, University of Tennessee, Knoxville, TN 37996, United States
Department of Biochemistry & Molecular Biophysics, Kansas State University, Manhattan, KS 66506, United States
Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri, Columbia, MO 65211, United States
Department of Technology, São Paulo State University, Jaboticabal, São Paulo, Brazil
Department of Biological Chemistry and Crop Protection, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United Kingdom
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada

 r  t  i  c  l e  i  n  f  o

rticle history:
eceived 22 April 2016
eceived in revised form 4 July 2016
ccepted 7 July 2016
vailable online 9 July 2016

eywords:
ndoplasmic reticulum
lycerol-3-phosphate acyltransferase
elianthus annuus
ass spectrometry

a  b  s  t  r  a  c  t

In oil crops,  triacylglycerol  biosynthesis  is  an important  metabolic  pathway  in which  glycerol-3-
phosphate  acyltransferase  (GPAT)  performs  the  first acylation  step.  Mass  spectrometry  analysis  of
developing  sunflower  (Helianthus  annuus)  seed  membrane  fractions  identified  an  abundant  GPAT,
HaGPAT9  isoform  1, with  a N-terminal  peptide  that  possessed  two  phosphorylated  residues  with  possi-
ble  regulatory  function.  HaGPAT9-1  belongs  to a broad  eukaryotic  GPAT  family,  similar  to  mammalian
GPAT3,  and  it represents  one  of  the  two  sunflower  GPAT9  isoforms,  sharing  90%  identity  with  HaGPAT9-2.
Both  sunflower  genes  are  expressed  during  seed  development  and  in vegetative  tissues,  with  HaGPAT9-1
transcripts  accumulating  at relatively  higher  levels  than  those  for HaGPAT9-2.  Green  fluorescent  pro-
tein  tagging  of  HaGPAT9-1  confirmed  its subcellular  accumulation  in  the endoplasmic  reticulum.  Despite
their  overall  sequence  similarities,  the  two  sunflower  isoforms  displayed  significant  differences  in their
riacylglycerol
east

enzymatic  activities.  For  instance,  HaGPAT9-1  possesses  in vivo  GPAT  activity  that  rescues  the  lethal
phenotype  of  the  cmy228  yeast  strain,  while  in  vitro  assays  revealed  a preference  of  HaGPAT9-1  for
palmitoyl-,  oleoyl-  and  linoleoyl-CoAs  of one  order  of magnitude,  with  the highest  increase  in  yield  for
oleoyl- and  linoleoyl-CoAs.  By contrast,  no enzymatic  activity  could  be detected  for  HaGPAT9-2,  even
though  its over-expression  modified  the  TAG profile  of  yeast.

©  2016 Elsevier  Ireland  Ltd.  All  rights  reserved.
. Introduction
Glycerol-3-phosphate acyltransferases (GPAT; E.C. 2.3.1.15) are
nzymes that transfer the acyl moiety from an acyl-coenzyme A

Abbreviations: ACP, acyl carrier protein; ConA, Concanavalin A; DAF,
ays after flowering; ER, endoplasmic reticulum; FOA, fluoroorotic acid;
3P, glycerol-3-phosphate; GFP, green fluorescent protein; GPAT, glycerol-3-
hosphate acyltransferase; LPA, 1-acylglycerol-3-phosphate or lysophosphatidic
cid; LPAAT, 1-acylglycerol-3-phosphate acyltransferase; PDAT, phosphatidyl-
holine:diacylglycerol acyltransferase; TAG, triacylglycerol.
∗ Corresponding author.

E-mail address: mvc@ig.csic.es (M.  Venegas-Calerón).

ttp://dx.doi.org/10.1016/j.plantsci.2016.07.002
168-9452/© 2016 Elsevier Ireland Ltd. All rights reserved.
(CoA) donor (or acyl–acyl carrier protein [ACP] in plastids) to the
sn-1 position of a glycerol-3-phosphate (G3P) molecule, yielding 1-
acylglycerol-3-phosphate (or lysophosphatidic acid, LPA) [1]. This
first acylation step occurs slower than the second [2], limiting the
availability of LPA and producing a potential ‘bottleneck’ in the
flow of carbon into glycerolipids, as originally defined by Eugene
Kennedy [3].

It is now realized that GPAT-mediated regulation of glycerolipid
synthesis is more complex than previously thought. Considerable
effort has been dedicated to determine the major plant GPAT iso-

forms implicated in seed oil synthesis, leading to the identification
of GPAT sequences from three protein families in different plant
species [4–6]. The GPAT family homologue of GPAT9 (At5g60620)

dx.doi.org/10.1016/j.plantsci.2016.07.002
http://www.sciencedirect.com/science/journal/01689452
http://www.elsevier.com/locate/plantsci
http://crossmark.crossref.org/dialog/?doi=10.1016/j.plantsci.2016.07.002&domain=pdf
mailto:mvc@ig.csic.es
dx.doi.org/10.1016/j.plantsci.2016.07.002


M. Payá-Milans et al. / Plant Science 252 (2016) 42–52 43

Table  1
Strains and plasmids used in this study.

Yeast strains and plasmids Description Reference or source

S. cerevisiae
S288C MAT� SUC2 gal2 mal2 mel flo1 flo8-1 hap1 ho bio1 bio6 [14]
W303-1A MAT� {leu2-3112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15} [15]
gat1� MATa; his3�1; leu2�0; lys2�0;ura3�0; YKR067w:kanMX4 [16]
ale1� MATa; his3�1; leu2�0; met15�0;  ura3�0; YOR175c:kanMX4 [17]
cmy228 W303-1A; MAT�; gat1�:TRP1 gat2�:HIS3 [pGAL1:GAT1 URA3] [18]

Plasmids
p416(LEU2) Yeast vector for constitutive expression of proteins under the control of a GPD promoter [19]

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pGAL1(URA3):GAT1 Plasmid carried by cmy228 that drives gal
pUC18/NheI-mGFP pUC18-based expression vector that harbo
pYES2(URA3) Yeast expression vector for native express

rom Arabidopsis thaliana L. has been attributed a direct role in TAG
iosynthesis [7,8]. This enzyme is located in the endoplasmic retic-
lum (ER) and its protein sequence shares certain similarity with
ammalian GPAT3 and GPAT4, both of which are involved in lipid

torage in white adipose tissue of the liver and mammary glands
9].

There have been several attempts to purify plant GPATs from ER
ractions. For instance, partial purification of a solubilized GPAT
rom avocado mesocarp (Persea americana Mill.) was achieved
y Eccleston and Harwood [10] using affinity chromatography,
lthough the purified enzyme was very unstable. A GPAT from palm
allus (Elaeis guineensis Jacq.) was also partially purified using ion
xchange and molecular exclusion chromatography by Manaf and
arwood [11]. Based on this latter approach, Ruiz-Lopez et al. [12]
btained fractions from sunflower (Helianthus annuus L.) enriched
n microsomal GPAT activity, although the enzyme responsible for
hat activity was not successfully sequenced.

Common sunflower seeds accumulate high levels of TAGs that
re rich in oleic and linoleic acids [13]. Moreover, several mutant
unflower lines have been selected and bred to produce oils with
iverse properties and fatty acid content [13]. The study of the
enetics and biochemistry of oil biosynthesis in sunflower seeds not
nly provides a basic understanding of the underlying processes but
lso, it yields potential targets to customize the lipid composition
f sunflower oil in order to improve both oil quality and produc-
ion. As such, the GPAT activity in developing seed embryos was
haracterized, whereby the highest activity was measured in seeds
etween 15 and 20 days after flowering (DAF), showing specificity
owards palmitoyl-CoA, oleoyl-CoA and linoleoyl-CoA derivatives
12]. The results of this study reflected the fatty acid composition
t the sn-1 position of sunflower TAGs, supporting the involvement
f this activity in oil assembly. To complement this earlier study,
e report here the successful identification of the major GPAT iso-

orm present in developing sunflower seed membranes, as well
s its molecular and biochemical characterization. The role of this
nzyme in sunflower oil synthesis is discussed in view of these
ndings.

. Materials and methods

.1. Biological material and growth conditions

The wild-type CAS-6 sunflower (H. annuus) line (Sunflower Col-
ection of Instituto de la Grasa, CSIC, Spain) was grown as described
n Ruiz-Lopez et al. [12]. Root, hypocotyl, leaf tissues and seeds
t different days after flowering (DAF) from at least three plants

ere harvested for downstream analyses. S. cerevisiae strains were

rown at 22 ◦C in restricted SC medium supplemented with glu-
ose, galactose or raffinose (2%, w/v). Tobacco (Nicotiana tabacum
.) Bright Yellow-2 (BY-2) cells were cultured in suspension and
 inducible expression of GAT1 to confer viability [18]
GFP gene driven by the CaMV35S promoter [20]

 proteins regulated by galactose Invitrogen

prepared as described elsewhere [7]. The strains and plasmids used
in this study are described in Table 1.

For heterologous expression studies and complementation
assays, the PLATE transformation method [21] was  used to sepa-
rately introduce the plasmid constructs into the yeast strains. Yeast
transformants were grown at 30 ◦C overnight in liquid selective SC
medium (SC-U for pYES2 and SC-L for p416) supplemented with
2% galactose. Complementation assays were performed on 5 �L
aliquots of serial dilutions plated on various selective media and
incubated at 22 ◦C. Yeast transformed with the empty plasmids
were used as controls.

2.2. Cloning and expression of HaGPAT9s

Sunflower HaGPAT9 isoforms 1 and 2 were identified based on a
BLAST search of Helianthus sp. EST sequences using human GPAT3
as the query. Alignments from these ESTs revealed two complete
GPAT sequences in sunflower, which were amplified from 15 DAF
sunflower seed cDNA using specific primer pairs (HaGPAT9 [1,2]-
F/-R: Electronic Material Table S1). The sequences of these cDNAs
were confirmed (Secugen, Madrid, Spain) and complete sequences
for HaGPAT9-1 and 2 were deposited in GenBank under accession
numbers EF552845 and EF552846, respectively.

Full-length sequences were amplified using the
GPAT9 [1,2]pYES2-F/-R primer pairs (Table S1) and they were
cloned into the pYES2(URA3) galactose inducible vector (Invitro-
gen, Carlsbad, CA, USA). Alternatively, the GPAT9 [1,2]p416-F/-R
primer pairs were used for constitutive expression in the yeast
p416(LEU2) vector [19] (kindly provided by Dr Ana Rincón). Quan-
titative PCR was performed using cDNA from seeds and tissues
obtained at various DAF, as described in Sánchez-García et al.
[22], and using the qGPAT9 [1,2]-F/-R primers (Table S1) and the
corresponding constructs in the pYES2 plasmid.

2.3. Mass spectrometry analysis of sunflower seed proteins

Solubilized proteins from sunflower seed microsomal fractions
were analyzed on the LTQ-Orbitrap XL mass spectrometer (Meth-
ods S1). The resulting spectra were searched against a custom
sunflower database containing data from NCBI, TIGR and UniGene,
as well as from the sunflower acyltransferases cloned to date that
were added manually.

2.4. Confocal microscopy of GFP-tagged HaGPAT9-1 in tobacco
cell suspensions

The full-length open reading frame (ORF, minus the stop

codon) of HaGPAT9-1 was  amplified by PCR using GPAT9 1pUC18-
F/-R primers, designed with NheI restriction sites (Table S1).
Cloned products were inserted into the unique NheI restriction
site of pUC18/NheI-mGFP [20]. The resulting construct encoded



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aGPAT9-1 linked at its C terminus to the monomeric green flu-
rescent protein (GFP).

Tobacco cells were transiently transformed with the plasmid
ncoding HaGPAT9-1-GFP as described previously [7]. The cells
ere then fixed and stained during the final 20 min of incubation
ith Alexa Fluor 594 conjugated Concanavalin A (ConA) [23] at

 final concentration of 5 mg/mL  (Molecular probes, Eugene, OR,
SA). Confocal laser scanning microscopy (CLSM) images of the
Y-2 cells were acquired on a Leica DM RBE microscope using a
3 x Plan Apochromat oil-immersion objective, a TCS SP2 scanning
ead (Leica Microsystems, Germany) and the LEICA TCN NT soft-
are package (Version 2.61). GFP was excited at 488 nm with an

rgon laser and with an emission filter opening at 480–550 nm.
mages of the cells were deconvoluted, and adjusted for brightness
nd contrast using Northern Eclipse 5.0 software (Empix Imaging,
anada). The images are representative of >20 cells from at least
hree independent transformation experiments and they were gen-
rated using Adobe Photoshop CS (Adobe Systems, San Jose, CA,
SA).

.5. Computational analysis

Phosphorylation sites were predicted using the GPS 3.0 server
24]. Protein sequences were aligned using Clustal Omega (version
.2.1) from the EMBL-EBI server [25,26], applying the default set-
ings. Gene ontology (GO) assignments were made using Blast2GO
.2 [27] from their closest GO-annotated orthologs in the plant
R database using default settings. A phylogenetic tree was  con-

tructed from the alignment using the minimum evolution method
n the MEGA 5.0 program and with a bootstrap value of 1000
28]. The programs used to predict the subcellular localization of
unflower HaGPAT9-1 were TargetP1.1 (www.cbs.dtu.dk/services/
argetP/) and ChloroP1.1 (www.cbs.dtu.dk/services/ChloroP/). The
rogram used to identify target regions that could potentially be
isrupted by translational fusion to the GFP was TMHMM (www.
bs.dtu.dk/services/TMHMM-2.0/). The OCTOPUS program (http://
ctopus.cbr.su.se/) was used to identify putative transmembrane
egions. Statistical significance between control replicates and
amples was obtained using Student’s t-test with P < 0.05.

.6. Analysis of the TAG and fatty acid composition of yeast cells

gat1� yeast strain transformed with a pYES2 construct either
ontaining a GPAT or empty was grown in SC-U selective media (see
ethods S1 for details). Overnight cultures were used to inoculate

alactose-containing media to induce enzyme expression. After
4 h growth, cells were harvested, washed and dried under nitrogen
urrent. The cell pellet was resuspended in methanol and heated at
0 ◦C for 10 min. Then, total lipids were extracted (Methods S1) and
ubjected to TAG purification [29]. The molecular TAG species were
nalyzed by MS  as described in Hamilton et al. [30]. The data were
ormalized to dry cell weight using the internal standard tri15:0
Nu-Check Prep, Elysian, MN,  USA).

.7. Preparation of yeast microsomal fractions

Yeast cells transformed with different pYES2 constructs were
arvested from 50 mL  cultures, and they were resuspended in
.5 mL  of a lysis solution containing 50 mM HEPES [pH 7.0], 2 mM
DTA and 10% (v/v) glycerol. An equivalent volume of 0.4–0.6 mm
cid treated glass beads (Sigma-Aldrich, St. Louis, MO,  USA) were
dded to the cells and they were subjected to six cycles of 30 s

rinding in a MiniBeadbeater-8 (BioSpec, Bartlesville, OK, USA) and
ooled for 1 min  on ice. The homogenate was spun at 600 g for

 min  and the supernatant was collected into a clean tube. A fur-
her 0.5 mL  of the lysis solution was added and the cycles of lysis
ience 252 (2016) 42–52

were repeated, adding the new supernatant to the former. This
soluble fraction was  centrifuged at 1500g for 5 min, collected and
then centrifuged at 16,000 g for 15 min. The microsomal fraction
was precipitated at 22,000 g for 2 h and it was  then resuspended in
50 �L 100 mM phosphate buffer [pH 7.5] for immediate use. All the
fractionation steps were carried out at 0–4 ◦C.

2.8. GPAT assay

GPAT activity was measured on microsomes of gat1� trans-
formants using a modification of the protocol reported by
Ruiz-Lopez et al. [12]. The incubation mixture (final volume of
100 �L) contained 50 mM bis-tris propane [pH 7.0], 1.5 mM G3P
(Sigma-Aldrich), including 3700 Bq L-[U-14C]glycerol-3-phosphate
(American Radiolabeled Chemicals, St. Louis, MO, USA), 0.3 mM
acyl-CoA (Sigma-Aldrich) and 0.05% (w/v) BSA. The reaction was
started with the addition of 50 �g of microsomal proteins and it
ran for 15 min  at 30◦C unless otherwise specified. The reactions
were stopped by adding 100 �L of 0.15 M acetic acid and the lipids
were extracted and analyzed as described originally.

2.9. LPAAT assay

LPAAT activity was measured on microsomes isolated from
ale1� transformants. The reaction mixture (a final volume of
100 �L) contained 100 mM phosphate buffer [pH 7.5], 50 �M 1-
oleoyl [1-oleoyl-9,10-3H]LPA (3667 Bq) (Perkin Elmer, Waltham,
MA,  USA), 20 �M acyl-CoA (Sigma-Aldrich), 1 mM MgCl2, 1 mM
DTT and 50 �g of microsomal proteins. After incubating at 30◦C
for 5 min, the reactions were stopped by adding 100 �L 0.15 M
acetic acid, and the lipids were extracted by adding a further 500 �L
of chloroform/methanol (1:1, v/v) followed by gentle mixing and
spinning at 2000 g for 10 min. The lower chloroform phase con-
taining the extracted lipids was  recovered and the aqueous phase
was re-extracted with a further 250 �L of chloroform. The lower
phase recovered was  combined with that previously extracted, it
was evaporated under nitrogen at 36◦C and reconstituted in 50 �L
of the chloroform/methanol mixture (2:1, v/v). Subsequently, 25 �L
of the lipid suspension was  analyzed by thin layer chromatogra-
phy, as described by Ruiz-Lopez et al. [12], and radioactivity in the
remaining volume was quantified using Ecoscint scintillant fluid
(National Diagnostics, Atlanta, GA, USA) and a LS-6500 Multipur-
pose Scintillation Counter (Beckman Coulter, Brea, CA, USA).

3. Results

3.1. Cloning of two sunflower GPAT genes: a phylogenetic study

Based on the sequences of previously described microsomal
GPAT3 from mouse and GPAT9 from Arabidopsis, we performed a
sequence homology search of ESTs in Helianthus species, identify-
ing fragments of two putative GPAT genes. The coding sequences
for these two isoforms were successfully amplified from sunflower
seed cDNA and they encoded proteins of 371 amino acids with
a predicted molecular weight of 42.6 kDa. Blast searches at the
nucleotide level using each isoform as a query against closely
related species in the Asterid subclass only yielded results for iso-
form 2 (data not shown). Even though sunflower behaves as a
diploid, the presence of two isoforms is probably due to the ances-
tral polyploid origin of the Helianthus genus [31]. Also, more than
one isoform were observed in other hybrid or polyploid species

(e.g., Malus x domestica,  Sesamum indicum, Glycine max: Fig. 1).

Comparison of these HaGPAT9 proteins with other similar pro-
teins revealed 75–90% identity with those from flowering plants,
45–70% with those from more distant Viridiplantae species and

http://www.cbs.dtu.dk/services/TargetP/
http://www.cbs.dtu.dk/services/TargetP/
http://www.cbs.dtu.dk/services/TargetP/
http://www.cbs.dtu.dk/services/TargetP/
http://www.cbs.dtu.dk/services/TargetP/
http://www.cbs.dtu.dk/services/TargetP/
http://www.cbs.dtu.dk/services/TargetP/
http://www.cbs.dtu.dk/services/ChloroP/
http://www.cbs.dtu.dk/services/ChloroP/
http://www.cbs.dtu.dk/services/ChloroP/
http://www.cbs.dtu.dk/services/ChloroP/
http://www.cbs.dtu.dk/services/ChloroP/
http://www.cbs.dtu.dk/services/ChloroP/
http://www.cbs.dtu.dk/services/ChloroP/
http://www.cbs.dtu.dk/services/TMHMM-2.0/
http://www.cbs.dtu.dk/services/TMHMM-2.0/
http://www.cbs.dtu.dk/services/TMHMM-2.0/
http://www.cbs.dtu.dk/services/TMHMM-2.0/
http://www.cbs.dtu.dk/services/TMHMM-2.0/
http://www.cbs.dtu.dk/services/TMHMM-2.0/
http://www.cbs.dtu.dk/services/TMHMM-2.0/
http://www.cbs.dtu.dk/services/TMHMM-2.0/
http://www.cbs.dtu.dk/services/TMHMM-2.0/
http://octopus.cbr.su.se/
http://octopus.cbr.su.se/
http://octopus.cbr.su.se/
http://octopus.cbr.su.se/
http://octopus.cbr.su.se/
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M. Payá-Milans et al. / Plant Science 252 (2016) 42–52 45

Fig. 1. Clade representation of proteins homologous to HaGPAT9. Phylogeny reconstruction using the minimum evolution method. Bootstrap values based on 1000 replications
a opsis 

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re  shown at the nodes with values below 50% condensed. Two  LPAATs from Arabid

5–35% identity with similar GPAT proteins in animals. Pro-
ein alignment across various kingdoms revealed that 18% of the

esidues were identical (Fig. S2). Clustering of homologous GPAT
roteins yielded a tree consistent with the taxonomic relation-
hips among their source species (Fig. 1), a tree that was  rooted
sing a couple of LPAAT proteins from A. thaliana as the outgroup.
thaliana were used to root the tree.

All sequences belonging to Viridiplantae species clustered well
together (88% bootstrap), while separate clades contained the rest

of the taxonomic groups (rhodophyta, amoebozoa, opisthokonta
and vertebrates). The two sunflower isoforms are grouped together
in a same clade.



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.2. Proteomic analysis of seed microsomes

In this study, sunflower seed microsomal proteins were ana-
yzed using a combination of one-dimensional gel electrophoresis
ollowed by liquid chromatography-mass spectrometry. The sam-
le complexity was reduced by SDS-PAGE pre-fractioning prior to
rypsin digestion (Fig. S1). In addition to the sunflower sequences
ontained in the online databases, known sequences for acyltrans-
erases were included in the main search database and they were
lso used to generate a specific database for post-translational mod-
fications.

After applying a 1% false discovery rate (FDR) cut-off, this
pproach resulted in the identification of 4413 non-redundant
eptides representing 991 proteins. From these, the product of
he HaGPAT9-1 gene (GenBank accession EF552845) was the most
oteworthy. The corresponding peptides were derived from the
DS-PAGE band containing proteins in the range of 35–40 kDa (Fig.
1).

A further search against a custom database containing sunflower
cyltransferase sequences was carried out to find peptides with
ost-translational modifications. This search revealed the presence
f four HaGPAT9-1 peptides defined by 7 spectra (Table 2). Of
hese, two peptides were unmodified, one was carbamidomethy-
ated and the other contained two phosphorylated residues (Tyr21
nd Thr32). The Tyr21 residue is conserved among all the flowering
lant species analyzed (Fig. S2), whereas Thr32 was only found in

soform 1. No deamidated peptides were found.

.3. Evaluation of the relevant proteins in sunflower microsomes

The assignment of GO terms to the proteins revealed that most
f the involved biological processes were related to protein and
ucleic acid metabolism (Table S3). 39 sequences were associ-
ted to lipid metabolic process (GO: 0006629); at least 20 of them
articipate in de novo lipid biosynthetic pathways and 7 in lipid
egradation (Table 3). Amongst the enzymes involved in lipid syn-
hesis that were associated with the ER, two long chain acyl-CoA
ynthetases were found, as well as a delta-12 oleate desaturase
nd a phosphatidylcholine:diacylglycerol acyltransferase (PDAT).
here was a large representation of soluble proteins related with
ntraplastidial fatty acid biosynthesis, including: acetyl-CoA car-
oxylase subunits, malonyl-CoA:ACP transacylase, components of
he fatty acid synthase complexes, acyl-ACP thioesterases and
tearoyl-ACP desaturases. Moreover, many proteins involved in
ipid degradation and �-oxidation were also present in this fraction,
uch as an acyl-CoA synthetase, a delta(3,5)-delta(2,4)-dienoyl-CoA
somerase, acyl-CoA oxidases and 3-hydroxyacyl-CoA dehydroge-
ases (Table 3). A large number of the identified spectra belonged
o proteins implicated in protein metabolism (Table S2). Moreover,
1S globulin seed storage proteins were also detected due to the pH
onditions used for precipitation in the preparation of the micro-
omes [32].

.4. Subcellular localization of HaGPAT9-1

The subcellular localization of HaGPAT9-1 was  studied in
obacco BY-2 cells by confocal microscopy [33–35]. The transient
xpression of HaGPAT9-1-GFP (via biolistic bombardment) in an
ndividual BY-2 cell revealed a reticular distribution that was
imilar to the fluorescence attributable to the ER marker ConA,
ndicating that HaGPAT9-1 is an ER resident protein (Fig. 2, top
ow). Closer inspection of the images revealed that the distribu-

ion of the GFP-tagged protein was not entirely uniform throughout
he ConA-stained ER but rather, it was at times enriched in dis-
inct regions of the ER or, less frequently, localized to regions
hat were devoid of ConA (Fig. 2, bottom row; see solid and open
ience 252 (2016) 42–52

arrowheads). The presence of aggregated ER structures was also
conspicuous in HaGPAT9-1-GFP-transformed cells (Fig. 2, top row).
Similar alterations in ER morphology were not detected in neigh-
boring untransformed BY-2 cells, yet they have been reported in
plant cells ectopically expressing other GPATs or other lipid biosyn-
thetic enzymes [36–38,7], suggesting that they are a consequence
of altering the amount of lipids in the ER membrane.

3.5. Functional complementation assay in yeast

An assay of GPAT activity based on the complementation of
the cmy228 S. cerevisiae strain (Table 1) was  designed to evalu-
ate the in vivo functionality of the HaGPAT9 proteins identified
(Fig. 3). Yeasts possess two  GPAT genes, GAT1 and GAT2, and dis-
rupting either of these alone does not cause detectable growth
effects. By contrast, disruption of both genes together is lethal.
The yeast cmy228 strain is gat1�gat2� and it contains the vec-
tor pGAL1(URA3):GAT1 that drives galactose inducible expression
of GAT1, allowing these cells to survive in the presence of this sub-
strate. To determine whether the activity of either of the sunflower
GPATs could replace the missing activity in this yeast mutant, they
were introduced into an expression system under the regulatory
control of the constitutive GPD promoter (p416[LEU2]:HaGPAT9-
1 and 2) and used to transform cmy288 strain. The prototrophic
strain S288C and the auxotrophic strain W303-1A carrying the
empty p416(LEU2) vector were used as URA+ and URA- controls,
respectively.

The strains grown under control conditions of plasmid selection
and induction (Fig. 3A) behaved quite distinctly to those grown
on glucose, where the lethal phenotype of the cmy288 strain
(Fig. 3B) was overcome by expressing the product of HaGPAT9-1
gene. However, the application of 5-fluoroorotic acid (FOA) to URA
supplemented medium, which is lethal for URA+ cells, inhibited
the growth of any cell that had not lost the pGAL1 plasmid (Fig. 3C).
Therefore, the HaGPAT9 does not fully complement yeast GAT func-
tionality. Growth in media not selecting p416 (Fig. 3D) triggered the
loss of such plasmids carrying the heterologous enzymes, decreas-
ing the rate at which cells grew (compare with Fig. 3B).

3.6. Overexpression of sunflower GPAT9s modifies the TAG profile
in yeast

A second approach to indirectly investigate the enzymatic activ-
ity of HaGPAT9s was  to analyze the modifications induced in yeast
TAGs. Sunflower GPAT coding sequences were introduced into the
inducible pYES2 expression vector, which was  transformed into the
GPAT deficient gat1� yeast strain. After growth on selective and
inducible media the yeast cells were harvested, dried and weighted,
and their TAGs extracted, analyzing both the TAG profile on a mass
spectrometer and the TAG fatty acid composition.

After normalization of the TAG data with internal lipid stan-
dards and according to samples dry weight, the over-expression
of both heterologous enzymes revealed an increase in the total
amount of TAGs produced by the transformants relative to the con-
trols (Fig. 4). HaGPAT9-1 expression induced the accumulation of
most TAG species, a composition not displayed by its counterpart.
The most abundant TAG species contained either 48 or 50 carbon
atoms. Regarding the degree of unsaturation, TAGs containing less
than C48 were present in saturated species, although there was a

preference to contain one unsaturated fatty acid, whereas longer
TAGs possessed at least one unsaturated fatty acid. Additionally,
the differences in the accumulation of TAG species were consistent
with the fatty acid composition of TAGs in the three strains (Fig. 5).



M. Payá-Milans et al. / Plant Science 252 (2016) 42–52 47

Fig. 2. Localization of HaGPAT9-1 to the ER in tobacco BY-2 cells transiently transformed (via biolistic bombardment) with HaGPAT9-1-GFP and stained with the ER marker,
ConA.  The merged image shows the co-localization of HaGPAT9-1-GFP and ConA at the ER. The asterisk in the merged image highlights a region of aggregated ER. The boxes
represent the area of the cell shown at higher magnification in the bottom row. Solid and open arrowheads in the bottom row represent examples where HaGPAT9-1-GFP
appears to be localized to distinct regions of the ConA-stained ER and regions devoid of ConA, respectively. Scale bar = 10 �m.

SC-L + FOA + 2% Glc

SC-UL + 2% GlcSC-UL + 2% Gal

SC-U +  2% Glc

1      10-1 10-2 10-3 10-4 10-5

A

B

C

D

E

1      10-1 10-2 10-3 10-4 10-51      10-1 10-2 10-3 10-4 10-5

1      10-1 10-2 10-3 10-4 10-5

A

B

C

D

E

A

B

C

D

E

A

B

C

D

E

A B

C D

Fig. 3. Complementation assay of the double GPAT cmy228 mutant yeast strain with the two  HaGPAT9 isoforms. The strains are displayed in rows and the dilutions in the
columns. (A-D) The plate composition is specified at the bottom of each image: A, S. cerevisiae strain S288C; B, S. cerevisiae strain cmy228 p416(LEU2); C, S. cerevisiae strain
cmy228  p416(LEU2):HaGPAT9-1; D, S. cerevisiae strain cmy228 p416(LEU2):HaGPAT9-2; E, S. cerevisiae strain W303-1A p416(LEU2).



48 M. Payá-Milans et al. / Plant Science 252 (2016) 42–52

Table 2
The mass spectrometry peptides belonging to the HaGPAT9-1 protein.

Peptide sequence Best SEQUEST XCorr score Number of total spectra Variable modifications identified by spectrum Charge state

GAFELGATVCPIAIK 2.69 6 c10: Carbamidomethyl (+57.02) +2H
IFVDAFWNSR 2.05 6 +2H
LRDLLDISPTLTEAAGAIVDDSFTR 2.88 6 +2H
PNIEDYLPPDSIQQPHTK 2.62 1 y6: Phospho (+79.97), t17: Phospho (+79.97) +3H

Table 3
Proteins in developing sunflower seeds identified by mass spectrometry that are related to lipid metabolism.

Protein gb Peptides

Biosynthesis
Acetyl-CoA carboxylase carboxyltransferase GE494904 DLYTHLTPIQR, GGVLSHLSPFKPLK, KHEYPWPQDPDPNVK
Malonyl-CoA:acyl carrier protein transacylase GE514621 GQAMQEAADAAK, LRGQAMQEAADAAK, VPAAAELYNK
Beta-ketoacyl-ACP synthase III (KAS III) EF514400 DKMTGLAVEAAQK, ILDAVATRLEIPADR, IVDTSDEWISVR, LEVSNDDLSK,

MTGLAVEAAQK, NVLVIGADALSR
Beta-ketoacyl-ACP synthase III (KAS III) GE514912 ILTGNESLTGLAAEASLK, IVDTNDEWISAR
Ketoacyl-ACP reductase 1 ADV16374 GITANAIAPGFISSDMTAK, LGEDIEKNILK
Beta-hydroxyacyl-ACP dehydratase ADE06392 ENFVFAGVDK, ENFVFAGVDKVR, KPVVAGDTLVMR
Beta-hydroxyacyl-ACP dehydratase ADL60215 DNFFFAGIDK, FPAFPTVIDINQIR, FPFLLVDR, VIEYNPGVSAVAIK
Enoyl-ACP reductase 2 HM021138 AFIAGIADDNGYGWAIAK, AVSASSDTKPLPGLPVDLR
Beta-ketoacyl-ACP synthase I (KAS I) EF177175 ALEDADLGGDK, ALEDADLGGDKLSK, LLAGESGIGLIDR, LLAGESGIGLIDRFDASK,

RLDDCLR
Beta-ketoacyl-ACP synthase I (KAS I) DY910493 ALEHADLAADNR, KALEHADLAADNR
Beta-ketoacyl-ACP synthase II DQ835562 ALADAGISPSDSDEIDKSR, VFNDAIEALR
Stearoyl-ACP desaturase U70374 AKESVNVPFSWIFDR, ATFISHGNTAR, ESVNVPFSWIFDR, HGDLLHQYLYLSGR,

VADLTGLSGEGR
Stearoyl-ACP desaturase U91339 AKEGPSIPFSWIFDR, AQDYVCGLPSR, KAQDYVCGLPSR, LAQICGTIAADEK
Long  chain acyl-CoA synthetase 4-like DY912306 HGPYVWLTYK, QVYDKVIQVGNAIR
Long  chain acyl-CoA synthetase DY911733 AAIGSGLVAHGIPK, VRPDGTVGDYK
Delta-12 oleate desaturase U91341 ALRPVLGEYYR, FACHYVPTSPMYNER, FDKTPFYVAMWR, GALATVDRDYGVLNK,

HHSNTGSLERDEVFVPK, YFNNTVGR
Acyl-ACP thioesterase FATA1 AY078350 CYEVGINK, KLNLIWVTSR, SSGEGLELNR, TGVAVDVTEKR, VNDDIRDEYLIFCPK,

WVMMNSETR
Acyl-CoA synthetase GE500285 KVFITDSVPK, RIVAEHFLTR
Phospholipid/glycerol acyltransferase (CDS gene = “ER-GPAT1”) ABU88984 GAFELGATVCPIAIK, IFVDAFWNSR, LRDLLDISPTLTEAAGAIVDDSFTR,

PNIEDYLPPDSIQQPHTK
Phosphatidylcholine:diacylglycerol acyltransferase GE497124 AMDPGLLDSEILGLK, SIMNIGPAFLGIPK, TWDSIISLLPK

Degradation
3-hydroxyacyl-CoA dehydrogenase DY909264 LGLIDAIAPPQDLLK, TDKIGSLSEAR
3-hydroxyacyl-CoA dehyrogenase DY909099 FSGGFDINVFQK, LGLIDAIVPPQDLLK, VFNELVLSDTSK
Acetyl-CoA acetyltransferase GQ254017 TPMGDFLGSLSSLPATK, ILVTLLGVLR, LGSIAIQSALQR
Acetyl-CoA C-acyltransferase/3-ketoacyl-CoA thiolase GE513651 LNVHGGGVSLGHPLGCSGAR, LGLNVIAK

405 

646 

937 

3
a

g
p
s
a
e
t
t
i
a
1
i
i
s
t
i

l
m
i

Acyl-CoA dehydrogenase DY920
Acyl-CoA oxidase DY907
Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, mitochondrial-like DY916

.7. Glycerol-3-phosphate and lysophosphatidate acyltransferase
ctivities of the HaGPAT9 proteins

The GPAT assays carried out on microsomal preparations of the
at1� yeast strain carrying the empty plasmid (control cells) dis-
layed reduced background activity. After over-expressing both
unflower isoforms, only the construct carrying HaGPAT9-1 induced
n increase in GPAT activity. The influence of reaction time was
valuated by running the assays for 15 or 30 min  (Fig. 6A) and as
he activity was 60% higher after the shorter 15 min  reaction time,
his was used in the following assays. The substrate specificity was
nvestigated using different acyl-CoA derivatives and the strongest
ctivity was evident with 16:0-CoA, followed by 18:2-CoA and
8:1-CoA (Fig. 6B). Compared to control, activity of yeast express-

ng HaGPAT9-1 with 16:0-CoA experienced a two-fold significant
ncrease while it raised to three-fold with the unsaturated sub-
trates. Only weak activity was displayed towards 18:0-CoA, close
o the limits of detection. No activity was detected for HaGPAT9-2
soform.
The LPAAT activity of GPATs was also evaluated using 1-oleoyl
ysophosphatidic acid as the acceptor and different acyl-CoAs. In

ost cases the expression of the GPATs in yeast did not significantly
ncrease the background LPAAT activity of the yeast microsomes
LGALNIAGGTIK, SWYFNHPALDVSK, VEGGWVIEGQKR
MANLVANDPAFEK, VSTHAVVYAR
LPGIVGFGNAMELALTAR, HMQDAITSIEK

(Fig. 6C), and a small but a significant increase in LPAAT activity
was only measured when HaGPAT9-1 was expressed and 18:1-CoA
or 18:2-CoA were the acyl donors. By contrast, the LPAAT activity
associated with expression of the second isoform was  lower than
that of the controls.

3.8. HaGPAT9 expression

The expression of microsomal GPATs was studied in developing
sunflower seeds and vegetative tissues by RT-qPCR. HaGPAT9-1 is
more actively transcribed than its homologue in most of the tissues
analyzed (Fig. 7A), and the expression of this isoform was  upregu-
lated at the initial phases of embryo development and at the late
stage of seed maturation. In addition, this isoform was strongly
expressed in leaves. The HaGPAT9-2 gene exhibited a very different
profile and it was generally expressed more weakly than its homo-
logue. This isoform was only upregulated at early and mid-stages
of seed development, as well as in vegetative tissues.
4. Discussion

TAGs accumulate during sunflower seed maturation to reach
values up to 40–50% of the total seed weight. The first acylation



M. Payá-Milans et al. / Plant Sc

Fig. 4. TAG molecular species in yeast over-expressing a sunflower HaGPAT9 and
the empty vector control. Values are the mean ± SD of three replicates. The asterisks
indicate statistically significant differences (P < 0.05) compared to the control.

F
a
i

r
a
fi
t
i
f
p
s
o

s
I
b
c
p

ig. 5. Fatty acid distribution in TAGs of yeast over-expressing a sunflower HaGPAT9
nd  the empty vector control. Values are the mean ± SD of three replicates. The aster-
sks indicate statistically significant differences (P < 0.05) compared to the control.

eaction to produce these TAGs is carried out by sn-1 GPAT enzymes
lthough in an initial search no such candidate proteins were identi-
ed in sunflower [12]. Later, 60 Kd sunflower GPATs were detected
hat were associated to a plant specific sn-2 GPAT family, GPATs 1–8
n Arabidopsis, involved in cutin/suberin biosynthesis [5]. No GPAT
rom this family was identified in our mass spectrometry analysis. A
rotein recently associated with TAG biosynthesis is the Arabidop-
is GPAT9 [7,8] and here, we provide an extensive characterization
f its counterpart in sunflower.

We  have cloned two GPAT genes from developing sunflower
eeds designated HaGPAT9-1 and -2 after the Arabidopsis gene.

soform 1 was the enzyme identified by proteomics in the mem-
ranes of embryos at the onset of the oil accumulation. This isoform
ontains some conserved blocks when aligned with homologous
roteins, indicating the probable fragments that define the active
ience 252 (2016) 42–52 49

site (e.g., the HX4D motif present in many acyltransferases) [39]
and substrate binding regions (Fig. S2). However, examination of
their amino acid sequences showed that both sunflower isoforms
accumulate changes in relation to their homologues.

A phylogenetic study showed a broad spectrum of taxonomic
groups that have proteins similar to this GPAT, suggesting an
ancient origin (at least in early eukaryotes) before they evolved into
the current kingdoms. The disposition of clades in the tree is coher-
ent with the group taxonomy, as partially reflected in the plant
clade. Investigation of the genome evolution in those plant species
carrying multiple isoforms of this enzyme points to hybridization
and changes in ploidy as the probable sources of the duplica-
tions. In view of our results, multiple isoforms do not appear to
imply enzyme redundancy. In this regard, the presence of multiple
GPAT proteins is frequent in nature, with the most representative
example of multiple gene duplication and functional diversifica-
tion depicted by the Arabidopsis GPAT1-8 family [5]. Conversely,
GPAT9 has a single functional isoform, recently revealed by mutant
analysis [8]. It is noteworthy that some eukaryotic clades do not
contain any GPAT9 homologues, the most relevant belonging to
Fungi kingdom. A Blast search only returned a few sequences
sharing moderate similarity, none of which belonged to the yeast
Dikarya sub-kingdom (Fig. S3). Instead, yeast enzymes with GPAT
activity, Gat1p and Gat2p [16], are more similar to a plant GPAT
family involved in polyester synthesis [40]. Furthermore, they not
only possess GPAT but also dihydroxyacetone phosphate acyltrans-
ferase (DHAPAT) activity, improving their functionality. In general,
GPATs exhibit a high degree of redundancy, not only due to the
presence of GPAT activity in various protein families but also, due
to the multiplication and evolution of distinct family members.
The fungal case is a clear example of how the redundant GPAT
activity present in most organisms produces viable organisms with
improved competitive capacity even after the loss of one isoform.

To identify GPATs involved in lipid biosynthesis and consid-
ering that these acyltransferases are membrane bound proteins,
membrane protein fractions from developing sunflower seeds (15
DAF) were analyzed by mass spectrometry. The only GPAT pro-
tein found in this fraction was  HaGPAT9-1 (GenBank accession
EF552845), which was unequivocally identified as the peptides
detected accounted for 68 of the 371 amino acids of the full length
sequence (18% coverage). Moreover, all them corresponded to
the HaGPAT9-1 isoform despite the high similarity with its close
homologue HaGPAT9-2 (90% amino acid identity, GenBank acces-
sion EF552846). Phosphoresidue analysis revealed the presence
of a peptide with two  phosphorylated sites at the N-terminus
of the protein (Tyr21 and Thr32), suggesting that its enzyme
activity or subcellular localization could be modified by specific
kinases. The Tyr21 residue is conserved among the homologues
in all the flowering plant species analyzed, suggesting a regu-
latory role of this amino acid. By contrast, Thr32 is unique to
HaGPAT9-1 and its function is not yet known. Interestingly, the
Gly32 residue in HaGPAT9-2 is also highly conserved in other
homologues, suggesting functional divergence in the regulation of
the two HaGPAT9 isoforms. The activity of yeast GPATs is regu-
lated post-translationally by phosphorylation of the C-terminus,
although it is not clear whether phosphorylation regulates GPAT
localization [41]. Our results support the potential phosphoryla-
tion of HaGPAT9-1 under physiological conditions, suggesting an
analogous regulatory mechanism.

At 15 DAF, both embryo growth and the accumulation of stor-
age oil by the seed are very active, as reflected by the complexity of
the enzymes determined by mass spectrometry that are involved

in biological functions like protein synthesis and fate, metabolism
and energy. The proteomic analysis of sunflower seed mem-
branes identified enzymes related to lipid metabolism other than
GPATs. For example, the identified PDAT belongs to an alternative



50 M. Payá-Milans et al. / Plant Science 252 (2016) 42–52

Fig. 6. GPAT enzyme assays in function of (A) time and (B) acyl-CoA substrate. (C) LPAAT assay as a function of the acyl-CoA substrate. Data are the mean of three replicates
with  SD error bars. The asterisks indicate statistically significant differences (P < 0.05) compared to the control.

Fig. 7. Expression of the HaGPAT9-1 (white columns) and -2 (light grey columns) genes in developing seeds and vegetative tissues of Helianthus annuus, and of GPAT9 from
A ys aft
A yos; 6
a s ± SD

p
p
a
a
s
s
o
t
D

m

rabidopsis thaliana. (A) Expression in sunflower determined by RT-qPCR: DAF, da
rabidopsis stages: 3, mid  globular to early heart embryos; 4, early to late heart embr
nd  10, green cotyledons embryos. The values in panel A represent the mean value

athway of TAG biosynthesis that involve the transesterification of
hosphatidylcholine (PC) and diacylglycerol (DAG) to yield lyso-PC
nd TAG. Previous research demonstrated that both diacylglycerol
cyltransferase (DAGAT) and PDAT contribute to TAG synthesis in
unflower seeds, and although DAGAT is more active, it has a lower
ubstrate affinity than PDAT [42]. Our finding highlights the interest
n this alternative pathway for TAG synthesis. Nonetheless, iden-
ification of other enzymes in the Kennedy pathway, LPAAT and

AGAT, is still missing.

Considering that the method used to obtain the microso-
al  fraction did not exclude organelle membranes, the proteins
er flowering. (B) Expression in Arabidopsis from microarrays of Schmid et al. [48].
, mid  to late torpedo embryos; 8, walking-stick to early curled cotyledons embryos;

 of three independent samples.

detected were from various subcellular localizations. Thus, many
plastidial enzymes related to fatty acid synthesis were found,
such as the stearoyl-ACP desaturase that was  among the most
abundant proteins in the microsomal fraction (Table S2) and
that is strongly expressed in sunflower embryos [13]. Also,
the first step in fatty acid synthesis is represented by the
alpha subunit of the acetyl-CoA carboxylase complex (sunflower
EST GE494904). However, not only enzymes related to lipid

biosynthesis were sequenced in microsomes but also those
involved in beta-oxidation, suggesting the development of peroxi-
somes for eventual seed germination. Despite the good coverage of



lant Sc

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M. Payá-Milans et al. / P

he activities in the lipid biosynthesis pathway, this method did not
apture proteins represented by enzymes either at a low abundance
r for which sequences have not yet been cloned in H. annuus.

The HaGPAT9-1 enzyme was clearly located in the ER and based
n the variation in fluorescence intensity, this enzyme is more
bundant in the perinuclear ER than in the cortical membrane when
ver-expressed in tobacco cells. Moreover, the ER morphology of
ells producing the heterologous protein is altered, forming mem-
rane aggregates in accordance with the altered ER morphology in
ells expressing mammalian GPAT3 [43] or A. thaliana GPAT9, and
ven GPAT8 [7]. Similar results were obtained after overexpression
f a human LPAAT [44]. Also, the accumulation of lipid interme-
iates may  recruit interactors by generating microdomains [45].
hen, the prominent globular ER structure is probably formed due
o: (i) the accumulation of a high proportion of acyltransferases;
ii) their activity altering the membrane properties; and/or (iii), the
ncrease in membrane destabilizing lipids. Moreover, the recurrent
lteration of the ER after an enrichment of acyltransferase activ-
ty also suggests the regulated formation of a possible lipogenetic
tructure.

In vivo functional assays were based on the complementation
f lethality that the disruption of both yeast GAT genes causes in
he cmy228 strain. Constitutive expression of HaGPAT9-1 allowed
east to grow without inducing GAT1 expression. Full complemen-
ation would have allowed yeast cells to lose the pGAL1(URA3)
lasmid carrying GAT1 and then grow under FOA negative selec-
ion. As indicated above, yeast GPATs belong to a different GPAT
amily and they exhibit GPAT/DHAPAT activities. Thus, HaGPAT9-

 activity complements the cmy228 strain under certain growth
onditions, although GPAT alone does not allow cell survival. Nev-
rtheless, this study reveals that HaGPAT9-1 encodes an enzyme
ith GPAT activity while that of its counterpart could not be deter-
ined. The TAG content and composition in yeast was also altered
hen sunflower GPATs were expressed, reflected by an increase in

he total amount of TAGs as well as an enrichment in 16:0 and 16:1
atty acids. Hence, both enzymes are active in the yeast heterolo-
ous system and their consequences are reflected in the alteration
f yeast lipids. In this regard, the significant modifications detected
n the strain over-expressing HaGPAT9-1 suggest enzyme selectiv-
ty towards saturated fatty acids. On the contrary, the HaGPAT9-2
nzyme does not change the TAG composition.

GPAT in vitro selectivity was studied in microsomal fractions
rom a yeast strain with reduced GPAT activity, the gat1� mutant.

icrosomes were incubated with radiolabeled G3P and differ-
nt acyl-CoAs, and the products were a mixture of LPA combined
ith lipids derived from this in the downstream TAG biosynthetic
athway. Thus, the total radioactivity in the products of LPA, phos-
hatidic acid (PA), diacylglycerol (DAG) and TAG were quantified.
ccordingly, only HaGPAT9-1 activity was detected in vitro, with
igh specificity towards 16:0-CoA and 18:2-CoA, lower specificity
owards 18:1-CoA and very low for 18:0-CoA. This activity is con-
istent with previous measurements of GPAT activity in sunflower
eed microsomes [12] and with the TAG composition of sunflower,

 species in which palmitic acid predominates at the sn-1 position.
he high GPAT specificity towards palmitoyl-CoA is also consistent
ith the increase in TAG species with a palmitoyl moiety at the sn-1
osition in yeasts transformed with HaGPAT9-1.

In light of the conservation of sequence motifs among acyl-
ransferases, the HaGPAT9s were tested for LPAAT activity. Indeed,
he length of these enzymes is similar to that of plant LPAATs,

uch shorter than the plant GPATs previously characterized [5].
e used the ale1� mutant reduced in lysophospholipid acyltrans-
erase activity as the host strain in these assays. HaGPAT9-1 strain
ad weak yet significant LPAAT activity with oleoyl- and linoleoyl-
oA substrates. However, direct implication of HaGPAT9-1 to the
hange in LPAAT activity could not be verified. Interestingly, the
ience 252 (2016) 42–52 51

LPAAT activity of membranes carrying HaGPAT9-2 was  significantly
reduced with the stearoyl-CoA substrate. Thus, although the iso-
form 2 did not utilize G3P or sn-1-LPA as acyl acceptors, it is an
enzyme that potentially competes for acyl-CoA substrates in a
yet unknown mechanism. Our results are in agreement with the
enzyme activity in microsomes from developing siliques of Ara-
bidopsis GPAT9 knock-down line [8], which was  reduced in GPAT
activity but not LPAAT activity.

HaGPAT9-1 is strongly expressed at the onset of TAG accumula-
tion in seeds and leaves; the pattern observed during seed filling
responds to the variations on the oil accumulation rate described
previously [46], suggesting that HaGPAT9-1 is involved in oil depo-
sition in sunflower seeds. By contrast, HaGPAT9-2 was strongly
expressed in vegetative tissues with only short periods of activation
in oil accumulating seeds. The first upregulation at 12 DAF coin-
cides with the end of the pericarp growth period while a second
upregulation at 21 DAF occurs when the embryo is actively devel-
oping [47]. This differs from the Arabidopsis GPAT9 transcripts [48]
that remain at similar levels in all the represented tissues and that
are slightly downregulated at early stages of embryo development,
suggesting constitutive expression of this gene. Also, a GPAT9-like
transcript was  found by RNA-sequencing of castor bean (Ricinus
communis) that is expressed in all tissues, and especially in the
developing seed endosperm [49], being the most abundant GPAT
transcripts in the endosperm. The analysis of the sunflower genes
performed here suggests that HaGPAT9-1, which is present in all
tissues, carries the ancestral GPAT9 activity, and there is certain
a functional divergence in this species revealed by the activity of
HaGPAT9-2.

Funding

This work was supported by the “Ministerio de Economia y Com-
petitividad” and FEDER [AGL2014-53537-R and JAE-CSIC to M.P-M].

Acknowledgments

We thank A. González-Callejas and B. Lopez-Cordero for their
skillful technical assistance. We  also thank Dr. Ana Rincón for kindly
providing the p416 (LEU) plasmid.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.plantsci.2016.07.
002.

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