Planta (2019) 249:333–350

https://doi.org/10.1007/s00425-018-3003-x

ORIGINAL ARTICLE

Genetic control of fatty acid composition in coconut (Cocos nucifera),

African oil palm (Elaeis guineensis), and date palm (Phoenix dactylifera)
Yong Xiao1 · Wei Xia2   · Annaliese S. Mason4 · Zengying Cao3 · Haikuo Fan1 · Bo Zhang2 · Jinlan Zhang2 ·
Zilong Ma3 · Ming Peng3 · Dongyi Huang2

Received: 14 May 2018 / Accepted: 3 September 2018 / Published online: 7 September 2018
© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
Main conclusion  Predominant gene isoforms and expression bias in lipid metabolism pathways are highly conserved
between oil-producing Arecaceae crop species coconut and oil palm, but diverge in non-oil-producing species date
palm.

Abstract  Coconut (Cocos nucifera), African oil palm (Elaeis guineensis) and date palm (Phoenix dactylifera) are three major
crop species in the Arecaceae family for which genome sequences have recently become available. Coconut and African oil
palm both store oil in their endosperms, while date palm fruits contain very little oil. We analyzed fatty acid composition in
three coconut tissues (leaf, endosperm and embryo) and in two African oil palm tissues (leaf and mesocarp), and identified
806, 840 and 848 lipid-related genes in 22 lipid metabolism pathways from the coconut, African oil palm and date palm
genomes, respectively. The majority of lipid-related genes were highly homologous and retained in homologous segments
between the three species. Genes involved in the conversion of pyruvate to fatty acid had a five-to-sixfold higher expression
in the coconut endosperm and oil palm mesocarp than in the leaf or embryo tissues based on Fragments Per Kilobase of
transcript per Million mapped reads values. A close evolutionary relationship between predominant gene isoforms and high
conservation of gene expression bias in the lipid and carbohydrate gene metabolism pathways was observed for the two oil-
producing species coconut and oil palm, differing from that of date palm, a non-oil-producing species. Our results elucidate
the similarities and differences in lipid metabolism between the three major Arecaceae crop species, providing important
information for physiology studies as well as breeding for fatty acid composition and oil content in these crops.

Keywords  Expression bias · Homologous segment · Lipid-related genes · Oil composition · Predominant gene isoforms

Abbreviations FPKM Fragments Per kb per Million reads

ACP Acyl carrier protein HAD Hydroxyacyl-ACP dehydratase
CALO Caleosins KAR Ketoacyl-ACP reductase
DGAT​ Diacylglycerol acyltransferase KAS Ketoacyl-ACP synthase
FATA(B) Acyl-ACP thioesterase A(B) LPAAT​ Lysophosphatidic acid acyltransferase
OBO Oil-body oleosins
PDAT Phospholipid:diacylglycerol acyltransferase
Electronic supplementary material  The online version of this
article (https​://doi.org/10.1007/s0042​5-018-3003-x) contains PDHC Pyruvate dehydrogenase complex
supplementary material, which is available to authorized users.

* Wei Xia
saizjxiawei@hainu.edu.cn; saizjxiawei@126.com
1
Coconut Research Institute, CATAS, Wenchang 571339,
Hainan, People’s Republic of China
2
Hainan Key Laboratory for Sustainable Utilization
of Tropical Bioresources, Institute of Tropical Agriculture
and Forestry, Hainan University, Haikou 570228,
People’s Republic of China
3
MOA Key Laboratory of Tropical Crop Biology and Genetic
Resources Utilization, Institute of Tropical Bioscience
and Biotechnology, CATAS, Haikou 571101, Hainan,
People’s Republic of China
4
Department of Plant Breeding, IFZ Research Centre
for Biosystems, Land Use and Nutrition, Justus
Liebig University Giessen, Heinrich‑Buff‑Ring 26‑32,
35392 Giessen, Germany

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Vol.:(0123456789)

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SAD Stearoyl-ACP desaturase
STERO Steroleosins
TAG​ Triacylglycerol
WPA Week post-anthesis
Introduction
Major crops date palm (Phoenix dactylifera), African oil
palm (Elaeis guineensis) and coconut palm (Cocos nucif-
era) are the only three species with sequenced genomes in
the family Arecaceae (Palmaceae). The date palm genome
was sequenced first in 2011 (Al-Dous et al. 2011), with
an improved version released in 2013 (Al-Mssallem et al.
2013), while an African oil palm genome assembly was
released in 2013 (Singh et al. 2013), and the coconut palm
genome was released 2017 (Xiao et al. 2017). One of the
major points of interest for comparison between these spe-
cies, as well as of importance for breeding purposes, is
their different oil production profiles. Coconut kernels have
approximately 63.1% oil content in copra (Lee et al. 2015).
A noticeable feature of coconut oil is that approximately
50% is lauric acid (­ C12:0), which is a type of medium-chain
length fatty acid (MCFA) (Laureles et al. 2002). African oil
palm also contains 50% lauric acid in its kernel oil (Dus-
sert et al. 2013) and has the highest oil content (90% of
dry weight) in its mesocarp reported for any plant tissue,
while date palm produces almost no oil in the mesocarp
of the fruits, but triacylglycerol (TAG) with different fatty
acids (mostly oleic acid) in the seeds (Alshahib and Marshall
2003; Bourgis et al. 2011). A number of genes differentially
expressed in coconut endosperm have been identified by
suppression subtractive hybridization (Liang et al. 2014).
However, no systematic analysis and comparison of lipid-
related genes between these three related species has been
performed, and detailed information about gene copy num-
bers and expression divergence in oil-producing tissues is
lacking for these crops.
RNA sequencing (RNA-seq) is widely used to study func-
tional gene expression profiles (Marioni et al. 2008). Tran-
scriptome analysis based on deep sequencing of expressed
sequence tags allows quantitative comparisons of gene
expression rates in different tissues, and even across multiple
species. In the Arecaceae, RNA-seq has been used to com-
pare the African oil palm and date palm transcriptomes dur-
ing mesocarp development, and synthesis of fatty acids and
supply of pyruvate in the plastid, rather than acyl assembly
TAG, was found to be the major factor controlling oil stor-
age in the oil palm mesocarp (Bourgis et al. 2011). RNA-seq
has been applied to examine the transcriptional basis of lipid
and carotenoid metabolism during the development of oil
palm mesocarp (Tranbarger et al. 2011). Lipid biosynthesis
has also been assessed in oil palm using transcriptomes of
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Planta (2019) 249:333–350
three different fruit and seed tissues that differ in oil content
and fatty acid composition (Dussert et al. 2013). Compara-
tive deep transcriptional profiling has also been conducted
for the four unrelated oilseeds Ricinus communis, Brassica
napus, Euonymus alatus and Tropaeolum majus, which dif-
fer in their oil storage tissues (Troncoso-Ponce et al. 2011).
Most of the enzymatic steps in lipid biosynthesis have been
defined at the molecular level based on lipid metabolism
work in Arabidopsis thaliana (Li-Beisson et al. 2013). With
the release of the coconut genome sequence and increased
availability of coconut transcriptome data, comparison of
lipid-related genes between coconut and related species Afri-
can oil palm and date palm could provide important infor-
mation for the identification of genes involved in regulating
quantitative and qualitative variation in lipid components.
In this study, we measured the fatty acid components and
total fatty acid content in coconut leaves, coconut endosperm
(four different developmental stages), and coconut embryo,
as well as in oil palm leaves and mesocarp tissues, and sys-
tematically identified genes related to lipid and carbohydrate
metabolism in the coconut, African oil palm and date palm
genomes based on orthology to genes known to belong to
lipid and carbohydrate pathways in A. thaliana. We com-
pared the gene number, location, evolutionary relationship,
and expression patterns of genes involved in fatty acid syn-
thesis, TAG accumulation and provision of precursors for
these pathways in the three Arecaceae species. Our study
elucidates the evolutionary relationships between the lipid
and carbohydrate metabolism pathways in the three species,
as well as providing clues to the divergent lipid accumula-
tion observed between tissues and species, providing a road-
map for further genetics and breeding studies in these major
fruit and oil crops.
Materials and methods
Sample collection for endosperm, embryo/
mesocarp and leaf tissues from coconut and African
oil palm
Coconut endosperm tissues were collected at 30, 36, 42 and
47 weeks post-anthesis (WPA). The embryo tissues were
collected from 52-week-old coconut fruits. All tissues and
coconut leaves were obtained from a 10-year-old “Hainan
Tall” cultivar plant in the Coconut Garden at Wenchang,
Hainan, China. The mesocarp tissues of African oil palm
were collected from 35-week-old oil palm fruits. African
oil palm leaves and mesocarp tissues were collected from a
5-year-old oil palm plant (a hybrid of a thick-shelled Afri-
can oil palm—dura and a thin-shelled African oil palm—
pisifera) grown in the oil palm germplasm nursery at the
Coconut Research Institute in Wenchang, Hainan, China.
Planta (2019) 249:333–350 335
Each sample collection was done with three biological rep-
licates. All samples were flash frozen and stored at − 80 °C
until needed for oil extraction, and four different develop-
mental stages of coconut endosperm tissues were also used
for RNA extraction.
Fatty acid extraction and quantification
Fatty acid extractions and analyses were performed in trip-
licate (from three different extractions) for each tissue and
each stage of development. Chloroform–methanol (2:1, v/v)
was used as a solvent for total lipid extraction following a
modified Folch method (Laffargue et al. 2007). Methyl ester-
ification was carried out by adding 1 ml of 14% BF3-metha-
nol solvent with incubation at 80 °C for 15 min. Dried sam-
ples were dissolved in 1 ml of n-hexane. Fatty acid methyl
esters were analyzed using gas chromatography (Agilent
7890A, column: Agilent DB-23, 30 m × 250 μm × 0.25 μm),
with peak area assessed using the default software pro-
vided (Agilent). Quantified heptadecanoic acid–methyl
ester (2 mg/ml) was used as an internal standard for lipid
quantification. TAG and fatty acid components in the coco-
nut leaf, endosperm and embryo, and in the oil palm leaf
and mesocarp, were calculated from the internal standard
using the formulas: (1) total fatty acids = (total area of all
measured fatty acid peaks × quantity of heptadecanoic
acid-methyl ester)/(peak area of heptadecanoic acid-methyl
ester × quantity of the sample); and (2) relative fatty acid
percentage = peak area of specific fatty acid/total area of all
measured fatty acid peaks.
Identification and characterization of lipid‑related
genes in C. nucifera, E. guineensis, and P. dactylifera
Predicted gene models for the coconut palm genome
were derived from Xiao et al. (2017), and three transcrip-
tome datasets for coconut leaf (SRR1273180), embryo
(SRR1265939) and endosperm (SRR1273070) tissues
generated by Huang et al. (2014) were downloaded from
the National Center for Biotechnology Information (NCBI;
https​://www.ncbi.nlm.nih.gov/). The genome sequences
and predicted gene models for African oil palm and date
palm were downloaded from NCBI, and all sequence read
archives (SRAs) used in this study are listed in Table S1.
The protein-coding genes for coconut, oil palm and date
palm were annotated with a translated BLAST using The
Arabidopsis Information Resource (TAIR) v8 database
(E value cutoff 1­ 0−10). Lipid-related genes were identi-
fied based on information available at http://arali​p.plant​
biolo​gy.msu.edu/. Genes putatively involved in lipid and
carbohydrate metabolism in coconut, oil palm and date
palm were selected based on information from Bourgis
et al. (2011) and Troncoso-Ponce et al. (2011). Gene fam-
ily classification was undertaken based on the annotated
protein information: genes classified as belonging to the
same family were either orthologous to A. thaliana genes
which had highly homologous amino acid sequences, or
were orthologous to the same A. thaliana gene. Gene pro-
moter regions of lipid-related genes from coconut palm,
African oil palm and date palm were analyzed and char-
acterized using the software program “TSSP”. Sequences
up to 2 kb upstream from the ATG start codons for lipid-
related genes were extracted and used for promoter analy-
sis; upstream sequences shorter than 500 bp were removed
from analysis. The phylogenetic tree was constructed by
the use of a neighbor-joining method in MEGA7 (Kumar
et al. 2016). Deduced amino acid sequences for the ana-
lyzed genes were aligned within MEGA7, and the aligned
results were manually checked before use. Bootstrapping
with 1000 replicates was used to establish the confidence
limits of the tree branches within the relationship dendro-
gram. Default program parameters were used.
RNA extraction and reverse transcription–real‑time
PCR assays
Total RNA was extracted from the coconut endosperm tis-
sues according to the Palmae RNA extraction method of
Xiao et al. (2012). The remaining DNA was removed by
RNase-free DNase treatment according to the manufactur-
er’s instructions (Promega). Total RNA concentration and
purity was determined using a Nanodrop ND-1000 spec-
trophotometer (Nanodrop Technologies). For each sam-
ple, first strand cDNA was synthesized according to the
manufacturer’s instructions (RevertAid First Strand cDNA
Synthesis Kit, Thermo Scientific). Real-time PCR was per-
formed following a standard SYBR Premix Ex TaqTM kit
(Roche) protocol in 96-well optical plates (Axygen) with
a final volume of 10 μl. The reactions were incubated in
0.2 ml tubes of an ABI 7900HT machine as follows: 95 °C
for 30 s, then 40 cycles of the program (95 °C for 5 s,
55 °C for 15 s and 68 °C for 20 s).
A set of 12 genes was selected for the RT-qPCR assay:
primer information for these genes is listed in Table S2.
After collecting the qPCR data, Ct values were con-
verted to relative quantities based on the formula: − 2deltCt
(deltCt = each corresponding Ct value—minimum Ct
value). The sample with the maximum expression level
(the minimum Ct value) was used as a calibrator and was
set to a value of 1. The ubiquitin-conjugating enzyme E2
10 (UBC10, accession No. GABT01019813), previously
identified as a stably expressed gene in endosperm tissue
(Xia et al. 2013), was used to normalize the gene expres-
sion from the qPCR data.
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336
Genomic context and homology
between lipid‑containing gene regions
between the three palm species
All protein-coding genes from coconut, African oil palm and
date palm were aligned using BLAST (Altschul et al. 1990)
against databases containing all protein-coding genes from
the three palm species with a cutoff of 1e-5. The BLAST
results were processed using the software MCScanX (Wang
et al. 2012). Homologous segments were defined as genomic
sequences containing a minimum of five homologous genes
in common with another genomic block from another palm
species. Here, only homologous blocks containing at least
one lipid gene were considered, although the blocks con-
tained other homologous non-lipid genes. All pairwise
homologous segments containing lipid-related genes were
identified and listed in Table S3.
Variation in lipid‑related gene expression
across coconut transcriptome data sets
The high-throughput RNA sequence reads were mapped
to the gene models and genomic sequences for the three
species’ genomes using bowtie2 (Langmead and Salzberg
2012), and the expression data for genes in the transcrip-
tomes derived from library sequencing of single end reads
were normalized using the RPKM method (Reads Per kb
per Million reads) (Mortazavi et al. 2008). Transcriptomes
derived from library sequencing with paired end reads were
normalized using the FPKM method (Fragments Per kb per
Million reads) (Trapnell et al. 2010).
Results
Lipid accumulation in coconut endosperm, embryo
and leaf tissues and African oil palm leaf tissue
To compare the major fatty acid composition across coco-
nut endosperm, embryo and leaf tissues, seven fatty acid
components were measured in the three tissue types. In the
four developmental stages of the coconut endosperm, the
percentage of lauric acid (C12:0) increased steadily across
the 7–11 month period, from an initial 7.7% to a final 49.6%.
During the same time period, palmitic acid (C16:0) con-
tent decreased from 24 to 8%, linoleic acid (C18:2) content
decreased from 29 to 5%, and linolenic acid (C18:3) con-
tent decreased from 24 to 3% (Fig. 1a). Fatty acid composi-
tion differed between the coconut endosperm and the leaf
and embryo tissues: the major fatty acids in the leaf were
linolenic acid (49%), palmitic acid (24%), and linoleic acid
(20%), and the major fatty acid components in the embryo
were linoleic acid (39%), palmitic acid (33%), and oleic acid
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Planta (2019) 249:333–350
(29%) (Fig. 1b). The total fatty acid content of the three tis-
sues was also quite different: the leaf fatty acid content was
about 0.7%; fresh mature embryo tissue had a total fatty acid
content of about 2%; the endosperm had an fatty acid content
of about 40% within its mature tissues (copra). In African oil
palm leaf tissue, very similar results were found compared to
coconut leaf tissue: 0.7% fatty acid with high linolenic acid
(55%) and palmitic acid (28%) (Fig. 1b).
Lipid‑related genes in the coconut palm, African oil
palm, and date palm genomes
Predicted genes (28 039 protein-coding genes) in the
coconut genome sequence (deposited in NCBI database:
SRA539146 with the project accession PRJNA374600 for
C. nucifera genome) were annotated by BLAST against the
Arabidopsis protein database (TAIR10): 806 genes were
orthologs of 418 genes involved in acyl-lipid metabolism
in Arabidopsis (E value cutoff < 10−10). The gene models
for African oil palm and date palm were also annotated by
BLAST against the Arabidopsis protein database: 840 and
848 lipid-related genes were annotated for African oil palm
and date palm, respectively (Table 1). A total of 418, 435,
and 432 lipid-related genes in Arabidopsis had correspond-
ing orthologs in the coconut, African oil palm, and date
palm genomes, respectively. For 495 lipid-related genes in
Arabidopsis from the three species, there were 77, 60, and 63
lipid-related genes where no corresponding orthologs could
be identified in the current coconut palm, African oil palm,
and date palm genome reference assemblies (Table 1 and
Table S4a). The copy number of annotated lipid-related gene
orthologs ranged from 1 to 9 in coconut palm, 1 to 14 in
African oil palm, and 1 to 11 in date palm. Two orthologous
genes were most commonly found (30–32% of Arabidopsis
genes across the three species), followed by one ortholog
(24–25% of genes) and then three orthologs (18–23% of
genes), with 19–22% of Arabidopsis genes having four-to-
seven orthologs across the three species (Table S5). Based
on lipid biosynthesis and biochemical pathways defined at
the molecular level in Arabidopsis, lipid-related genes from
the three species could be categorized as belonging to 22
lipid metabolism pathways (Table 1). Among these lipid
metabolism pathways, cuticular wax synthesis, lipase, and
lipid signaling had the largest number of genes in the three
species, also corresponding to the lipid pathways with the
largest number of genes in Arabidopsis (80, 73, and 102
genes, respectively).
The majority of lipid-related genes homologous between
coconut, African oil palm, and date palm were located in
homologous genomic segments (Table 2). Similar num-
bers of genes were homologous between species pairs: 793
and 777 lipid-related genes in coconut aligned with 775
genes in African oil palm and 758 genes in date palm (E
Planta (2019) 249:333–350 337
Fig. 1  Fatty acid (FA) composition in coconut endosperm, leaves,
and embryos and in African oil palm leaves and embryo. a FA com-
position in the coconut (Cn) endosperm at 30, 36, 42, and 47 WPA. b
FA composition in coconut leaves and embryo and African oil palm
(Eg) leaf and mesocarp, along with the oil contents. The relative con-
values < 1e-50), while 798 genes in African oil palm were
homologous with 790 genes in date palm. Analysis of pro-
moter regions 2 kb upstream of the translation start site ATG
for the lipid-related genes showed that 80–84% of homol-
ogous gene pairs had the same putative promoter motifs,
but with noticeable divergence in promoter motifs between
species for some genes (Table 2). Most lipid-related genes
were located in scaffolds containing more than five protein-
coding genes in coconut (74.5%, 601/806), African oil palm
(80.3%, 666/829), and date palm (87.5%. 702/802). Using
the criterion of > 5 homologous genes per block to define
homologous segments between species, 485 homologous
segments were identified between coconut palm and Afri-
can oil palm, and 422 homologous segments were identi-
fied between coconut palm and date palm. Moreover, 72.8%
(438/601) and 61.9% (372/601) of genes in coconut palm
were located in homologous segments with African oil palm
and date palm, respectively. A schematic display of homolo-
gous segments across coconut palm, oil palm, and date palm
is shown in Fig. S1. The homologous segments compared
between the three species include oil palm chromosome 1
tents of seven fatty acids are presented: caprylic acid (C8:0), capric
acid (C10:0), lauric acid (C12:0), myristic acid (C14:0), palmitic acid
(C16:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid
(C18:3)
(NC_025993.1), 38 coconut scaffolds, and 49 date palm
scaffolds.
Transcriptional patterns for enzymes involved
in the conversion of pyruvate to fatty acid
Three coconut sequence read archives (SRAs), eight Afri-
can oil palm SRAs (one leaf tissue, five mesocarp tissues,
and two kernel tissues), and six date palm SRAs (one
leaf tissue and five mesocarp tissues) were downloaded
from NCBI and used for lipid-related gene expression
analysis in leaf, endosperm, mesocarp, and embryo tis-
sues (Table S1). A total of 41 enzymes in Arabidopsis,
62 enzymes in coconut, 60 enzymes in African oil palm,
and 56 enzymes in date palm catalyze the conversion of
pyruvate to fatty acid; in coconut, 32 of these related were
located in homologous segments with African oil palm or
date palm (Table S4a and Table S6). More of these genes
were highly expressed (FPKM ≥ 30) in coconut (41 out
of 62) and oil palm (42 out of 60) than in date palm (11
out of 56) (Tables S4a and S6). Half of these enzymes
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338 Planta (2019) 249:333–350

Table 1  Numbers of genes related to lipid metabolism in the three palm species coconut (Cocos nucifera), oil palm (Elaeis guineensis), and date
palm (Phoenix dactylifera) relative to Arabidopsis thaliana 
Lipid-related pathway Cocos nucifera Elaeis Phoenix dac- Arabidopsis
guineensis tylifera
Orthologs to Total
palmae

Aliphatic suberin synthesis 43 39 36 23 34

Aromatic suberin synthesis 18 17 21 6 8
Beta-oxidation 28 28 27 20 25
Cuticular wax synthesis 104 108 94 80 167
Cutin synthesis 39 32 31 22 28
Eukaryotic phospholipid synthesis 59 70 63 35 45
Fatty acid elongation and cuticular wax synthesis 44 43 44 25 26
Galactolipid degradation 10 10 6 6 7
GDSL 5 6 6 3 106
Lipase 126 117 108 73 271
Lipid acyl hydrolase 12 15 13 9 11
Lipid signaling 180 168 164 102 142
Lipid trafficking 11 10 14 6 6
Miscellaneous lipid-related 67 71 70 42 74
Mitochondrial fatty acid and lipoic acid synthesis 13 15 15 9 13
Mitochondrial phospholipid synthesis 20 24 25 17 10
Phospholipase 76 69 66 40 92
Plastidial fatty acid synthesis 71 68 63 36 48
Plastidial glycerolipid, galactolipid and sulfolipid synthesis 80 74 77 43 52
Sphingolipid synthesis 49 54 59 24 28
TAG degradation 39 43 39 26 35
TAG synthesis 69 90 80 42 68
Total 806 840 848 495 885

The lipid-related genes in the palm species were annotated by a translated BLAST against the Arabidopsis Information Resource v8 database (E
value cutoff ­10−10)

had relatively conserved gene copy numbers across the

four species, including pyruvate dehydrogenase complex
(PDHC), acyl carrier protein (ACP), stearoyl-ACP desatu-
Table 2  Homologous segments (containing a minimum of five rases (SAD), and acyl-ACP thioesterase A (FATA) genes
homologous genes) containing lipid-related genes between Cocos (Table S6). For the four enzyme subunits of PDHC (E1-α
nucifera (Cn), Elaeis guineensis (Eg), and Phoenix dactylifera (Pd) and -β, E2, and E3), all four species contained one-to-three
Gene category Number of genes gene copies of E1-α, E-β, and E3, while coconut palm and
African oil palm had six and four gene copies of enzyme
Cn/Ega Cn/Pd Eg/Pd
subunit E2 relative to two in Arabidopsis and three in date
Homologous genes 793/775b 777/758 798/790 palm, although only three copies of these E2 genes were
Genes with Pro* 667/632 654/607 649/636 highly expressed (FPKM ≥ 30) (Table S4a).
Pro in both 625/600 607/577 602/599 There are six stearoyl-acyl carrier protein desaturase
Genes without Pro 49/47 47/42 53/47 (SAD) genes in Arabidopsis, of which only three genes have
Homologous genes in 438/472 372/393 503/494 orthologs in coconut palm, African oil palm, and date palm;
homologous segments albeit in two-to-four copies for orthologs of AT2G43710 and
Homologous segments 485 422 560 AT3G02630 and a total of seven to eight SAD genes. Gene
Pro*: putative promoter motif (TATA box or enhancer) copy numbers in the three species for acetyl–CoA carboxy-
a
 The species’ genomes were aligned to identify homologous seg- lase (ACCase) complex, ketoacyl-ACP synthase (KASI, -II,
ments containing lipid-related genes and -III), and acyl-ACP thioesterase B (FATB) were also
b
 Number of genes present in the corresponding species about two-to-five times that of Arabidopsis.

13
Planta (2019) 249:333–350 339
The FPKM values for 17 plastidial proteins that are
involved in conversion of pyruvate to fatty acids were, on
average, five-to-sixfold higher in the endosperm of coco-
nut palm and the mesocarp of oil palm than in the leaf or
embryo of coconut palm or in the leaf of oil palm, respec-
tively (Table S4a and Fig. 2). ACP, ketoacyl-ACP reduc-
tase (KAR), and hydroxyacyl-ACP dehydratase (HAD)
were the top three enzymes with high FPKM values in the
coconut endosperm, while ACP, KAR, and PDHC were the
top three enzymes in the oil palm mesocarp (Fig. 2). The
acetyl-CoA precursor required for de novo fatty acid syn-
thesis is provided by the activity of PDHC, and the FPKM
values of PDHC were five-to-ten times higher in tissues with
higher oil content (i.e., the endosperm of coconut palm and
the mesocarp of oil palm) than in the other tissue types.
Although the FPKM value of PDHC is higher in date palm
mesocarp than in leaf tissues, it was ten times lower than in
the endosperm of coconut palm and the mesocarp (and even
leaf) of oil palm.
Fig. 2  Gene expression values for enzymes involved in fatty acid and
glycerolipid synthesis in leaf, embryo, endosperm, and mesocarp tis-
sues for coconut palm, oil palm, and date palm. The expression level
The ACCase complex catalyzes the carboxylation of
acetyl-CoA to malonyl-CoA, and its FPKM value was high-
est in the oil palm mesocarp, followed by the coconut and
oil palm endosperms. Similar expression patterns of higher
FPKM values in the coconut endosperm and oil palm meso-
carp were also found for the rest of the enzymes which cata-
lyze the conversion of pyruvate to fatty acid, such as ACP,
KAR, ketoacyl-ACP synthase III (KAS III), and stearoyl-
ACP desaturase (SAD) genes (Table S4a, Fig. 2). Of the
three SAD genes in Arabidopsis with orthologs in the three
palm species (At2g43710, At1g43800, and At3g02630),
At2g43710 orthologs were most highly expressed in these
tissues (Table S1).
The two acyl-ACP thioesterases [acyl-ACP thioesterase
A (FatA) and acyl-ACP thioesterase B (FatB)] terminate
plastid fatty acid synthesis. There were two FATA​genes in
coconut palm and date palm and three FATA​genes in Afri-
can oil palm, with two copies of genes with FPKM ≥ 30
in coconut palm and African oil palm (Table S4a). The
of a gene family was calculated from the sum of the FPKM values for
isoforms and subunits
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340
FPKM value of FATA​was seven and twofold higher in the
coconut endosperm than in the coconut embryo and leaf,
respectively. The FPKM value of FATA​ was also tenfold
higher in oil palm mesocarp (23 weeks old) than in oil
palm leaf and higher in 15-week-old oil palm mesocarp
than in 15-week-old oil palm endosperm. No expression
was detected for EgFATA​—LOC109505499, which had a
truncated amino acid sequence, while LOC105048939 was
the most highly expressed EgFATA​. Based on amino acid
sequences, phylogenetic analysis for FATA​genes in the three
species showed that LOC105048939 (EgFatA) was the same
gene identified in Dussert et al. (2013) as belonging to the
same group as CCG012754 (CnFatA) and LOC103716139
(PdFatA) (Fig. S2). Coconut, oil palm, and date palm all
contained five FATB genes, although the EgFatB gene
(LOC109506086) in oil palm with a truncated amino acid
sequence was subsequently excluded from phylogenic anal-
ysis. The three FatB genes in oil palm mesocarp or ker-
nel tissues with high FPKM values were named EgFatB1
(LOC105049016), EgFatB2 (LOC105040257), and EgFatB3
(LOC105047747). Based on sequence similarity and the
phylogenic tree constructed from amino acid sequence
alignments (Fig. 3), the five corresponding FatB genes in
coconut were named after the oil palm FatB genes: CnFatB1
(CCG011598.1), CnFatB2-1 (CCG006479.1), CnFatB2-2
(CCG007799.1), and CnFatB3 (CCG019705.1), with one
new FatB gene named as CnFatB4 (CCG015192.1). Three
of the coconut FatB genes were highly expressed in more
than one analyzed tissue: CnFatB2-1 (leaf and embryo),
CnFatB2-2 (leaf, embryo, and endosperm), and CnFatB3
(embryo and endosperm). CnFatB4 clustered with EgFatB4
(LOC105049288) and PdFatB4 (LOC103705673) from
Fig. 3  Phylogenetic relationship between FatB genes in the three
palm species Cocos nucifera (Cn), Elaeis guineensis (Eg), and
Phoenix dactylifera (Pd), and the corresponding FPKM values for
13
Planta (2019) 249:333–350
date palm: all three genes had no or low expression across
all the analyzed tissues (Table S4a). Only one FatB gene
in date palm (PdFatB2-1; LOC103720846) had medium
expression levels in the leaf; the remaining four FatB genes
had no or low expression in leaf and mesocarp. Based on
the homologous genomic segment analysis, FatB2, FatB3,
and FatB4 were located in homologous regions and con-
served between the three species (Table S7 and Fig. 4). The
detailed evolutionary relationship between these genes was
displayed using phylogenic tree and homologous segment
comparisons (Figs. 3 and 4b). Genomic segments contain-
ing FatB2 also had high homology with genomic segments
containing FatB3 (Fig. 4b). Moreover, homologous seg-
ments containing CnFatB2-1/EgFatB2 had better collin-
earity than homologous segments containing CnFatB2-1/
EgFatB3: more homologous genes pairs were identified in
the CnFatB2-1/EgFatB2 homologous segments (Fig. 4a,
b). Homologous segments containing PdFatB2-2/EgFatB3
were also detected. No homologous segment containing
CnFatB2-2 was found in African oil palm, and the scaf-
fold where CnFatB2-2 was located in coconut was short
and contained only nine protein-coding genes (Table S7).
CnFatB2-2, which has no corresponding EgFatB gene, was
highly expressed in three tissues (FPKM > 30), but with
fourfold higher expression in the endosperm than in the leaf
and embryo, while both CnFatB3 and EgFatB3 were highly
expressed in coconut and oil palm endosperms. EgFatB1
was highly expressed in the oil palm endosperm, while
CnFatB1 and PdFatB1 were expressed at low levels or were
not expressed in the analyzed tissues.
Free fatty acids are esterified to CoA by long-chain
acyl-CoA synthetases (LACS) at the plastid envelope
expressed FatB genes at 15 WPA and 23 WPA, respectively. Grey
boxes around gene names indicate very low (FPKM < 5) or no expres-
sion across all tissue types assessed
Planta (2019) 249:333–350 341
Fig. 4  Schematic maps for homologous segments containing FatB
genes across the three palm species Cocos nucifera (Cn), Elaeis
guineensis (Eg), and Phoenix dactylifera (Pd). The colored lines indi-
cate homologous genes between species and lines in the same color
in a subfigure indicate genes present in the same homologous seg-
ment. a Segment of oil palm chromosome 3 (NC_025995.1, contain-
or endoplasmic reticulum. We identified 12, 11, and 13
LACS genes in coconut palm, oil palm, and date palm,
respectively. Of the nine LACS genes in Arabidopsis, the
AtLACS4 (At4g23850) and AtLACS9 (At1g77590) orthologs
in coconut palm and oil palm were highly expressed, and
the AtLACS3 (At1g64400) ortholog was additionally highly
expressed only in coconut palm (FPKM ≥ 100) (Table S1a).
The FPKM values of LACS9 ortholog in coconut were
similar in the leaf and endosperm tissues and lower in the
embryo, while the FPKM values of LACS9 ortholog were
much higher in the African oil palm mesocarp than in the
endosperm and leaf, and lower in both the leaf and mesocarp
of date palm (FPKM < 20). The two LACS9 genes from oil
palm and date palm were located in homologous segments
relative to coconut palm (Table S7).
ing EgFatB2) aligned with coconut and date palm scaffolds. b Seg-
ment of oil palm chromosome 6 (NC_025998.1, containing EgFatB3)
aligned with coconut and date palm scaffolds. c Segment of oil palm
chromosome 1 (NC_025993.1, containing EgFatB4) aligned with
coconut and date palm scaffolds
The expression profiles of 12 genes, including CnCT-α,
CnBCCP, CnKAS III, CnKAR, CnHAD, CnKAS II, CnKAS
I, CnSAD, CnFatA, and CnFatB, were detected in four devel-
opmental stages of coconut endosperm. The expression lev-
els of EgCT-α, EgBCCP, and EgKAS II in the mesocarp
increased greatly from 15 WPA to 19 WPA, and decreased
again at 23 WPA (Table S4a). Similar variation in expression
was detected in the coconut endosperm for CnCT-α, CnB-
CCP, CnKAS III, and CnKAR, with a fast rise in expression
in the 36 WPA endosperm but a decrease in the 42 WPA
or 47 WPA endosperm (Fig. S2). Moreover, the expression
levels of EgCT-α, EgBCCP, EgKAS II, and EgKAS III in oil
palm endosperm were decreased in the 15 WPA mesocarp.
For EgKAS I and EgHAD, sustained high expression levels
in the mesocarp were observed from 19 WPA to 23 WPA
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(Table S4a), while the expression levels for CnHAD and
CnKAS I showed no significant variation from 36 WPA to
42 WPA (Fig. S3). EgSAD genes showed high expression
(FPKM ≥ 700) across different mesocarp developmental
stages, but decreased expression levels from the 10 WPA
endosperm to the 15 WPA endosperm. Two CnSAD genes
(CCG025689.1 and CCG019622.1) and one CnFatA gene
(CCG012754.1) had sharply decreased expression from 30
WPA to the next three stages. However, two key CnFatB
genes (FPKM ≥ 100) had an increased gene expression dur-
ing endosperm development, with the highest expression
level at 47 WPA.
Similar expression levels for enzymes facilitating
synthesis of TAG from Acyl‑CoA in leaf, embryo,
and endosperm tissues
Acyl chains are assembled into TAG by a series of
membrane-associated reactions which include glycerol-
3-phosphate acyltransferases (GPAT), LPA acyltransferase
(LPAAT), phosphatidate phosphatase (PP), and diacylg-
lycerol acyltransferases (DGAT), among others. Moreover,
phospholipid:diacylglycerol acyltransferases (PDAT) cata-
lyze diacylglycerol (DAG) to form TAG as the final step in
TAG synthesis using phospholipids. In coconut and oil palm,
the expression level of genes involved in the conversion of
Acyl-CoA to TAG was lower when compared to the expres-
sion level of genes involved in the conversion of pyruvate
to fatty acid (Fig. 2). GPATs catalyze the first acylation (sn-
1) of glycerol-3-phosphate to yield lysophosphatidic acid
(LPA): AtGPAT9 (At5g60620) is the Arabidopsis protein
most similar to a mammalian GPAT important for TAG
synthesis (Cao et al. 2006; Gidda et al. 2009). One-to-two
AtGPAT9 orthologs were identified in each of the three palm
species: FPKM values for these genes were similar between
embryo and endosperm tissues in coconut and between leaf
and mesocarp tissues in oil palm, but oil palm had higher
expression of AtGPAT9 orthologs in 10 WPA endosperm
tissues (Table  S4a). Most other GPAT gene members
(GPAT1–GPAT8) were expressed at low levels, except for
GPAT3 which was highly expressed in leaf tissues.
LPA acyltransferases (LPAAT) catalyze the second acyla-
tion in de novo TAG assembly. Among the five Arabidopsis
isoforms of this enzyme family, AtLPAAT2 (At3g57650) is
the most highly expressed (Kim et al. 2005). Two-to-three
AtLPAAT1 or AtLPAAT2 orthologs were found in each of
coconut, oil palm, and date palm (Table S4a). Based on
the phylogenic analysis, these LPAAT1 genes were clus-
tered into class I and class II (Fig. 5a). CCG006531.1 was
excluded from phylogenic analysis as a result of incomplete
sequence. LPAAT1 genes in class II had low or no expres-
sion, while class I genes had higher expression levels in
endosperm tissue. LOC105035151, the EgLPAATB gene
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Planta (2019) 249:333–350
reported in Dussert et al. (2013), was expressed fourfold
higher in 15 WPA endosperm than in leaf and mesocarp
tissue. CCG001603.1, the same LPAAT​ gene identified
by Davies et  al. (1995) and characterized by Knutzon
et al. (1995, 1999), was highly expressed in the coconut
embryo (FPKM = 146.5) and endosperm (FPKM = 394.2)
(Fig. 5a). The remaining genes in class II had low expres-
sion levels in all tissues, except for CCG024643.2 which
had high expression in the leaf (FPKM = 50.3). The AtL-
PAAT2 orthologs were also clustered into two classes, and
genes in class II had low or no expression (Table S4a and
Fig. 5b). LOC105035151, belonging to class I and named as
EgLPAATA​in Dussert et al. (2013), was expressed fivefold
higher in 15 WPA endosperm than in the leaf and meso-
carp. Moreover, CCG006531.1 and CCG015599.1 showed
medium or high expression levels in three tissues, and had
twofold higher expression in endosperm than in leaf or
embryo tissues (Table S4a and Fig. 5b).
Diacylglycerol acyltransferases (DGAT) and PDAT cata-
lyze diacylglycerol (DAG) to form TAG as the final step in
TAG synthesis, using either acyl-CoAs or phospholipids,
respectively. Five-to-six DGAT isoforms were identified in
coconut (6), African oil palm (6), and date palm (5), includ-
ing orthologs for DGAT enzymes of type 1 (AtDGAT1)
and type 2 (AtDGAT2), and AtDAcT (wax synthase-like).
DGATs were more highly expressed in coconut endosperm
than in the leaf and embryo, especially for DGAT1 isoform
CCG007098.3 and DGAT2 isoform CCG026159.1 (Fig. 6
and Table  S4a). DGAT1 was three times more highly
expressed than DGAT2 in coconut endosperm, but DGAT1
and DGAT2 were close in expression in the coconut leaf and
embryo and in the three oil palm tissues (Table S4a). The
expression level of DGATs was lower in date palm tissues
than in coconut and African oil palm leaf tissues. The novel
acetyltransferase enzyme (DAcT) had a low expression level
in the three palm species. PDATs transfer the sn-2 acyl group
from phospholipids to DAG: AtPDAT1 orthologs were much
more highly expressed in date palm leaf than in the coco-
nut palm and oil palm tissues, while AtPDAT2 (At4g19860)
orthologs were more highly expressed in coconut leaf than
in coconut embryo/endosperm tissues, but were close in
expression between oil palm tissues (Fig. 6 and Table S3a).
PDATs in coconut palm were expressed at lower levels than
DGATs, but had similar expression in oil palm. The ortholog
of AtPDAT1 in date palm (LOC103708654) was very highly
expressed in leaf tissue (FPKM = 784.6).
Both coconut palm and African oil palm have high lauric
acid (C12:0) content in their endosperm tissues and high
linolenic acid (C18:3) in their leaf tissues, while African oil
palm has high palmitic acid (C16:0) and oleic acid (C18:1)
contents in mesocarp tissue. Oleate desaturase (FAD2) was
expressed at three times higher levels in coconut embryo
and endosperm compared to coconut leaf tissue, but FAD2
Planta (2019) 249:333–350 343

Fig. 5  Phylogenetic relationship between LPAAT​ genes in the three nut (Cocos nucifera) leaf, embryo and endosperm tissues, and in oil
palm species Cocos nucifera (Cn), Elaeis guineensis (Eg), and Phoe- palm (Elaeis guineensis) leaf; mesocarp 15 WPA, mesocarp 23 WPA,
nix dactylifera (Pd) and the corresponding FPKM values of lysophos- and endosperm 15 WPA
phatidic acid acyltransferase. a LPAAT1. b LPAAT2 genes in coco-

Fig. 6  FPKM values of diacylglycerol acyltransferases (DGAT1 and in oil palm (Elaeis guineensis) leaf; mesocarp 15 WPA, mesocarp 23
DGAT2) and phospholipid:diacylglycerol acyltransferases (PDAT) in WPA, and endosperm 15 WPA
coconut (Cocos nucifera) leaf, embryo and endosperm tissues, and

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was expressed more than 100-fold higher in African oil palm
mesocarp than in African oil palm leaf and embryo tissues
(Table S4a). Moreover, linoleate desaturase (AtFAD7 and
AtFAD8) orthologs showed 40-to-50-fold higher expression
in coconut and oil palm leaves than in other tissues, but were
only expressed at low levels in date palm (FPKM < 1).
Divergence in storage of triacylglycerol (TAG)
between endosperm, embryo, and mesocarp tissues
Triacylglycerols accumulate in oil bodies, which have a
TAG core surrounded by a phospholipid monolayer and
abundant amphipathic proteins. Three classes of pro-
teins were specifically expressed in seed-related tissues
(i.e. embryo and endosperm): oil-body oleosins (OBO),
steroleosins (STERO), and caleosins (CALO) (Fig. 7). Of
these three classes of proteins, oil-body oleosins (OBO)
were the protein family with the highest number of genes
differentially expressed between tissues. The orthologs of
AtOBO, AtSTERO, and AtCALO in coconut and oil palm
were expressed at 40-to-500-fold higher levels in embryo
and endosperm tissues than in leaf and mesocarp. CALO
expression was similar between the coconut endosperm
Fig. 7  FPKM values of oil-body oleosin (OBO), caleosin (CALO), steroleosin (STERO) and lipid droplet-associated proteins (LDAPs) in coco-
nut leaf, embryo and endosperm tissues, and in oil palm leaf; mesocarp 15 WPA, mesocarp 23 WPA, and endosperm 15 WPA
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Planta (2019) 249:333–350
and the oil palm endosperm, but OBO was expressed at
eightfold higher levels in coconut endosperm relative to oil
palm endosperm. The expression levels of steroleosins and
caleosins were four-to-tenfold higher in embryo tissue than
in endosperm tissue in coconut, and 5-to-50-fold higher in
oil palm endosperm than in oil palm leaf and mesocarp.
All orthologs of AtOBO, AtSTERO and AtCALO in date
palm were expressed at low levels (FPKM ≤ 10).
Oil palm mesocarp is an important oil storage tissue,
but only low expression levels of OBO, STERO, and CALO
were detected in this tissue type. Hence, another class of
lipid droplet-associated proteins called LDAPs, which
localize specifically to the lipid droplet surface within
plant cells, was analyzed, to see if LDAP expression was
compensating for OBO/STERO/CALO expression in oil
palm. Three orthologs of AtLDAP (AT3G05500) were
identified in the three palm species, and were expressed in
leaf, embryo, endosperm, and mesocarp tissues (Table S4a
and Fig. 7). LDAP expression values were similar between
the leaf and embryo tissues in the three palm species, but
were 20-to-30-fold higher in both the coconut endosperm
and oil palm mesocarp.
Planta (2019) 249:333–350 345

The majority of highly expressed lipid‑related gene
copies were orthologous between coconut and oil
palm
Gene families with multiple lipid-related gene paralogs
usually had only one-to-two paralogs which were highly
expressed in the coconut endosperm or oil palm mesocarp:
“predominant” genes. The majority of these predominant
highly expressed genes were also located in homologous
segments between the two species (Table S7). In five of
the seven multi-gene families belonging to the plastid fatty
acid synthesis pathway (PDHC, HAD, KAS I, KAR, ACP,
and SAD), predominant genes accounted for more than
86% of the total gene expression (Table 3; ACCase and
KASIII did not show this pattern). Nine out of ten pre-
dominant genes in this pathway were determined to be
in homologous segments between coconut and oil palm.
The Acyl-CoA-binding protein (ACBP) gene family had
9–12 copies in the three palm species, but orthologs of
AT1G31812 showed predominant expression in coconut
and oil palm (Table S4a).
The ortholog of AT3G54320—WRINKLED1 (WRI1)
is considered to be a key transcription factor associ-
ated with lipid synthesis in oil palm. We identified three
AT3G54320 orthologs in coconut, six in oil palm, and
four in date palm, none of which were expressed in the
leaf, coconut embryo, or date palm mesocarp (Table 3
and Fig. 8). The 13 WRI1 genes could be clustered into
three groups based on conserved amino acid sequences,
each of which showed distinct expression patterns (Fig. 8).
WRI1 group I was highly expressed in the coconut
endosperm and the oil palm endosperm/mesocarp, with
LOC105046121 expressed specifically in endosperm tis-
sue. WRI1 group II showed low expression in coconut

Table 3  Predominant gene copy (most highly expressed gene copy) in multi-gene families in the plastid fatty acid synthesis pathway in coconut
(Cocos nucifera: Cn) and oil palm (Elaeis guineensis: Eg)
Gene abbreviation Predominant Cn- FPKM in coconut Predominant Eg- FPKM in oil palm Gene in
isoform endosperm isoform homologous
Mesocarp at 15 Mesocarp at 23 segment
WPA WPA

PDH-E1α CCG009120.1 207.6 LOC105059287 130.5 1052.0 NA

PDH-E1β CCG003104.1 498.6 LOC105059550 208.1 1412.8 Yes
LTA2 CCG022878.1 105.8 LOC105035588 30.4 249.7 NA
LPD2 CCG016999.1 123.9 LOC105039925 53.7 362.2 No
PDHC 935.8/1083.3 (86%)a 422.6/485.3 (87%) 3076.8/3376.1
(91%)
CT-α CCG021872.1 220.7 LOC105054827 62.6 245.5 Yes
BCCP CCG016556.1 108.6 LOC105055979 40.9 124.8 NA
BC CCG016422.1 89.0 LOC105050891 46.7 510.7 Yes
ACCase 418.3/660.6 (63%) 150.1/194.4 (77%) 881.1/1208.9 (73%)
KASI CCG024029.1 152.9 LOC105040922 14.9 258.5 Yes
KASI CCG026381.1 212.3 LOC105053059 24.1 79.0 Yes
KASI 365.2/426.3 (86%) 38.9/61.3 (64%) 337.5/417.0 (81%)
KASIII CCG003289.1 140.4/216.6 (65%) LOC105056570 7.3/70.0 (10%) 212.8/290.3 (73%) Yes
KAR CCG006105.2 881.5/999.6 (88%) LOC105051367 212.6/237.0 (90%) 1064.3/1122.0 Yes
(95%)
HAD CCG001741.1 1985.3/2038.3 LOC105051936 51.6/69.0 (75%) 289.2/339.4 (85%) Yes
(97%)
ACP4 CCG000806.1 1251.6 LOC105057703 32.6 395.2 NA
ACP4 CCG027016.1 2075.3 LOC105045224 161.9 3125.4 Yes
ACP 3326.8/3836.7 194.5/210.0 (93%) 3520.6/3703.7
(87%) (95%)
FAB2 CCG005175.1 572.3 LOC105048931 334.9 532.1 NA
FAB2 CCG019622.1 2009.8 LOC105049670 372.0 1427.5 NA
SAD 2582.1/2966.6 706.9/715.3 (99%) 1959.6/2026.9
(87%) (97%)

NA not available because of the short scaffold length in the coconut palm genome assembly where the gene was located
a
 Number in bold before the slash is the FPKM sum for the predominant isoform, the number after is the FPKM sum for all isoforms, and the
number in brackets is the percentage ratio between the two FPKM values

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Fig. 8  Phylogenetic analyses of WRINKLED1 (WRI1) genes from
coconut, oil palm, and date palm, and the corresponding expression
FPKM values of WRI1 genes in coconut leaf, embryo, and endosperm
and oil palm endosperm, while WRI1 group III showed
no or low expression in all the analyzed tissues of the
three species.
Different expression patterns for genes involved
in providing carbon for fatty acid synthesis
via conversion of sucrose to pyruvate
Sucrose and glucose are the major sources of carbon in
maternal tissues. For the initial sucrose metabolism path-
way, coconut leaf tissue showed higher gene expression lev-
els of neutral invertase (N-INV), cell wall invertase (CW-
INV), and beta-fructosidase (vacuolar-INV) than coconut
endosperm and embryo tissues. This was especially noticea-
ble for vacuolar-INV which was expressed at 50-to-100-fold
higher levels in the leaf, but sucrose synthase (SuSy) was
highly expressed in all three coconut tissues (Table S4b).
In oil palm, the mesocarp tissue showed higher expres-
sion of N-INV, CW-INV, vacuolar-INV, and sucrose syn-
thase (SuSy), especially for CW-INV which was expressed
fivefold higher than in the leaf and 100-fold higher than in
the endosperm. CW-INV and SuSy showed fivefold higher
expression in the mesocarp than in the leaf for date palm,
but much lower expression than in the other two species
(Table S4b).
Glycolysis provides the large amounts of pyruvate
required for oil synthesis, and has eight steps occurring
in both the cytosol and plastid. In coconut endosperm and
embryo, genes involved in cytosol glycolysis were expressed
more than sevenfold higher than genes involved in plastid
glycolysis, but genes involved in cytosol and plastid gly-
colysis were expressed at similar levels in the coconut leaf
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Planta (2019) 249:333–350
tissues, and in oil palm leaf; mesocarp 15 WPA, mesocarp 23 WPA,
and endosperm 15 WPA. Grey boxes around gene names indicate
very low (FPKM < 5) or no expression across all tissue types assessed
(Table S4b). In oil palm, genes involved in cytosol glycolysis
were expressed two-to-threefold more than genes involved in
plastid glycolysis in leaf, mesocarp, and endosperm tissues,
and in the date palm mesocarp, genes involved in cytosol
glycosis were expressed 12-fold higher than genes involved
in plastid glycolysis. In general, genes involved in cytosol
glycolysis in coconut tissues had a higher expression val-
ues (5-to-100-fold) than genes involved in plastid glycoly-
sis, with the exceptions of fructose-bisphosphate aldolase
(FBA), phosphoglycerate kinase (PGK), and triosephosphate
isomerase (TPI), which showed balanced or higher expres-
sion in the leaf. Pyruvate kinase (PK), the gene involved in
the last catalyzing step of plastid glycolysis, had a similar
expression in the endosperm (Table S4b). In oil palm leaf
and mesocarp tissues, cytosol glycolysis genes were also
three-to-tenfold more highly expressed than plastid glyco-
lysis genes, with again the exception of FBA and PK. In the
coconut endosperm, PGK, phosphoenolpyruvate enolase
(ENO), and PK were two-to-threefold more highly expressed
than in coconut leaf and embryo, while FBA, ENO, and PK
were two-to-threefold more highly expressed in the oil palm
mesocarp than in the other two tissues (Table S4b).
ATP-dependent phosphofructokinase (PFK) catalyzes
fructose-6-phosphate to fructose-1,6-bisphosphate, a reac-
tion which is reversible by pyrophosphate-dependent phos-
phofructokinase (PFP) in the cytosol. The expression of
cytosol-PFK was higher than the expression of PFP and
plastid-PFK in the three coconut tissues as well as in the oil
palm mesocarp and endosperm.
The most striking differences with regarD to carbo-
hydrate pathways between the three palm species con-
cerned plastid transporters (Table  S4b and Fig. 9). The
Planta (2019) 249:333–350 347
Fig. 9  Gene expression for triose phosphate translocator (TPT), phos-
phate translocator (PPT1), and phosphate transporter 2 (GPT2) in
coconut (Cocos nucifera) leaf, embryo, and endosperm tissues, and
glucose-6-phosphate/phosphate transporter 2 (GPT2)
ortholog was the most highly expressed carbohydrate-
related gene in the oil palm mesocarp, with an expres-
sion level 17-to-30-fold higher than in the oil palm leaf
and endosperm, or in any of the analyzed tissues of date
palm and coconut palm (Table S4b). On the other hand,
the orthologs of phosphoenolpyruvate (PEP) transporter
PPT1 were highly expressed in the coconut endosperm, at
threefold higher levels than in the oil palm mesocarp, and
at sixfold higher levels than in the coconut embryo. PPT1
was lowly expressed in date palm. Moreover, triose phos-
phate translocator (TPT) showed 14–20 times higher expres-
sion levels in coconut leaf than in coconut endosperm and
embryo, which themselves had moderate-to-low expression
levels (FPKM ≤ 30). TPT also had higher expression in oil
palm leaf and mesocarp than in the oil palm endosperm.
Discussion
In this study, we analyzed fatty acid composition and total
fatty acid content in three coconut tissues (leaf, endosperm,
and embryo) and in two oil palm tissues (leaf and meso-
carp), and identified 806, 840, and 848 lipid-related genes
in 22 lipid metabolism pathways from the coconut, oil palm,
and date palm genomes, respectively. Subsequently, com-
parison of lipid-related genes between the palm species
showed that the majority of lipid-related genes in coconut
in oil palm (Elaeis guineensis) leaf; mesocarp 15 WPA, mesocarp 23
WPA, and endosperm 15 WPA
were highly homologous to genes in African oil palm and
date palm: 72.8 and 61.9% of lipid-related genes in coconut
palm were located in homologous segments with African
oil palm and date palm, respectively. For enzymes involved
in the conversion of pyruvate to fatty acid, more genes with
higher expression levels were detected in coconut and oil
palm than in date palm, and expression levels for these genes
were five-to-sixfold higher in the oil storage tissues of the
coconut endosperm and oil palm mesocarp than in the leaf
and embryo tissues. Moreover, the genes involved in conver-
sion of Acyl-CoA to TAG were expressed at lower levels
than genes involved in the conversion of pyruvate to fatty
acid in coconut and oil palm. These results suggested that
although the lipid-related genes from the three palm spe-
cies were highly conserved with respect to their sequences
and genomic locations, differences in expression levels of
genes related to synthesis of fatty acids, pyruvate supply, and
plastid-specific transport determine fatty acid production.
Close evolutionary relationships between highly expressed
gene copies in these pathways were identified between coco-
nut and oil palm, along with noticeable differences in FatB,
WRI1, and other gene copies. Comparison of genes involved
in lipid and carbohydrate metabolism in the palm species
suggested that the two oil-producing palm species have
highly conserved expression patterns for most orthologs,
although noticeable differences were observed for fatty acid
synthesis and TAG accumulation, as well as provision of
precursors for these two pathways. Our results suggest that
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348
despite the evolutionary divergence between the two spe-
cies, coconut and oil palm still use the same conserved lipid
biosynthesis pathways (down to the individual gene level)
to store oil in the endosperm (coconut) or fruit (oil palm).
A. thaliana acyl-lipid metabolism requires at least 120
enzymatic reactions and more than 600 genes to encode
the proteins and regulatory factors involved (Li-Beisson
et al. 2013). Gene alignment between Arabidopsis and the
three palm species indicated that 24–25% of the identified
lipid-related genes in the three palm species were single
copy, while 50–55% of lipid-related genes had two-to-three
orthologous genes corresponding to one Arabidopsis gene
(Table S5). Coconut palm and oil palm are important oil
crops and have a similar fatty acid composition in their
endosperms. For genes involved in the conversion of pyru-
vate, a single highly expressed copy for each lipid-related
gene family accounted for 70–90% of total gene expression,
and these genes tended to be located in homologous seg-
ments between coconut and oil palm (Table 3). High conser-
vation of promoter sequences, expression bias, and genomic
location was observed for lipid-related genes between the
three palm species, along with noticeable gene copy-number
variation (Table S6). Sequence conservation in promoter
regions was in accordance with the similar expression bias
observed for predominant gene isoforms between species.
Our analysis showed that date palm has conserved lipid-
related genes compared to coconut and African oil palm,
but the expression patterns for these lipid-related genes are
distinctly different, such that most of these genes have low
or no expression. For multiple gene families (e.g., FatB
and WRI1), evolutionarily conservation of genes with no
observed expression in the tissue types assessed in our study
between coconut palm and African oil palm was observed,
suggesting that subfunctionalization of these genes occurred
prior to speciation.
Analysis of gene expression profiles between the oil stor-
age tissues and the other tissues indicated that a similar gene
expression bias existed in the coconut endosperm relative to
the oil palm mesocarp (Fig. 2). A similar result for fatty acid
synthesis genes was reported in a transcriptomic comparison
between African oil palm fruit and date palm fruit (Bour-
gis et al. 2011). Although endosperm and embryo comprise
two different oil storage tissues in most species, the tran-
scriptional patterns of most genes in the plastidial fatty acid
synthesis pathway are generally similar between these two
tissue types (Troncoso-Ponce et al. 2011). For most enzymes
involved in TAG synthesis, we found no substantial differ-
ence between leaf and endosperm gene expression, except
for a few genes (such as DGAT, which is a key gene for TAG
synthesis) (Fig. 7).
The previous research focused on the high lauric acid
accumulation in coconut endosperm and oil palm endosperm
indicated that CnFatB and EgFatB genes are responsible for
13
Planta (2019) 249:333–350
the production of medium-chain and short-chain fatty acids
(Dussert et al. 2013; Jing et al. 2011; Yuan et al. 2017). RT-
qPCR analysis of CnFatA and CnFatB genes in our study
showed that the decrease of CnFatA gene expression and
the increase of CnFatB expression were accompanied by an
increase of medium-chain fatty acid content. The compari-
son of five FatB genes in the palm species showed that the
more highly expressed FatB copies were conserved between
species. However, noticeable divergence was observed for
CnFatB2-2 in coconut and PdFatB2-2 in date palm (Fig. 3),
which have no corresponding homologous genes in oil
palm and may play an important role in lipid biosynthesis
in the coconut endosperm (FPKM = 263.4), but having a
low expression level in data palm (Table S4a). The analysis
of homologous segments containing FatB genes suggested
that FatB2 and FatB3 may be derived from genomic segment
duplication, while CnFatB2-1, EgFatB2, and PdFatB2-1
were located in homologous segments between the three
palm species. The extra FatB2-2 gene copy may be derived
from the genomic segment containing FatB3. The multi-
gene family FatB also showed a complex expression pattern
in the three coconut tissues, although one which differed
to that observed in closely related species African oil palm
(Fig. 3).
The WRINKLED1 (WRI1) gene is considered to play
an important role in oil accumulation (Focks and Ben-
ning 1998; Cernac and Benning 2004; Bourgis et al. 2011;
Troncoso-Ponce et al. 2011; Dussert et al. 2013). Genes
found to be highly up-regulated during rapid accumulation
of seed oil were also found to be differentially expressed
between soybean accessions with high and low seed oil
contents (Bourgis et al. 2011; Troncoso-Ponce et al. 2011;
Zhang et al. 2016). In our study, three groups were identified
across the three palm species based on sequence conserva-
tion for WRI1 gene orthologs. The WRI1 ortholog group
I was highly expressed exclusively in oil palm mesocarp
tissues, the WRI1 ortholog group II was expressed in both
coconut and oil palm endosperm, and the WRI1 ortholog
group III showed low expression across all the tissues and
species (Fig. 8). These results support a major role of par-
ticular WRI1 orthologs in oil accumulation in coconut and
oil palm, and indicate that the comparative analysis of tran-
scriptome data for different oil storage tissues can identify
genes involved in oil accumulation and the determination of
fatty acid composition.
TAG is packaged in dynamic subcellular organelles
termed oil droplets (ODs) or oil bodies (Huang 1996). The
most abundant proteins’ coating seed ODs are oleosins,
and genetic studies in A. thaliana mutants have suggested
a key role for oleosins in preventing ODs from coalescing,
thereby maintaining ODs as small discrete entities to facil-
itate TAG mobilization and confer freezing tolerance dur-
ing post-germinative seedling growth (Siloto et al. 2006;
Planta (2019) 249:333–350 349
Shimada et al. 2008). Over-expression of PDAT1 increases
leaf TAG accumulation, leading to oil droplet overexpan-
sion through fusion, while co-expression of PDAT1 with
oleosin boosts leaf TAG content by up to 6.4% of the dry
weight without affecting membrane lipid composition and
plant growth (Fan et al. 2013). In avocado mesocarp tissue,
LDAP1 and LDAP2 proteins (homologous to At3g05500
in Arabidopsis) were associated with the surface of non-
seed lipid droplets, and were proposed to be involved in
the general process of binding and, perhaps, the stabili-
zation of lipid-rich particles in the cytosol of plant cells
(Horn et al. 2013). In the present study, OBO, CALO,
and STERO were specifically expressed in seed-related
tissues (i.e., embryo or endosperm) but rarely expressed
in the oil palm mesocarp, which has also been reported
by Tranbarger et al. (2011) and Dussert et al. (2013). This
study analyzed the expression of LDAPs, which showed
higher expression levels in both the coconut endosperm
and the oil palm mesocarp (Fig. 7). These results sug-
gest that the oil palm mesocarp does not require oleosin-
mediated stabilization, but that LDAPs are involved in
TAG accumulation in this tissue type instead, while both
oleosin-mediated stabilization and LDAPs may be active
in the coconut endosperm.
The comparison of carbohydrate metabolism between
coconut and oil palm showed distinct differences in the bal-
ance of glycolysis occurring in the plastid relative to in the
cytosol, as well as in plastid transporters which provide gly-
colytic substrates to the plastid by transport from the cytosol
(Table S4b and Fig. 9). The oil palm mesocarp had a high
expression of GPT2 and PPT1, which transport hexose P and
phosphoenolpyruvate (PEP) to the plastid from the cytosol,
accompanied by increased expression of plastid- fructose-
bisphosphate aldolase (FBA) relative to cytosol-FBA, and
plastid-pyruvate kinase (PK) relative to cytosol-PK (Bourgis
et al. 2011; Dussert et al. 2013). Coconut endosperm also
had a high expression of PPT1 accompanied by increased
expression of plastid-FBA relative to cytosol-FBA. These
data indicate that most pyruvate used for fatty synthesis in
coconut and in oil palm is generated from PEP in both the
plastid and the cytosol. Higher plastid/cytosol expression
ratios for FK and FBA along with higher expression of TPT
have previously been detected in B. napus embryos, suggest-
ing that B. napus may allow a greater proportion of hexose
to pyruvate flux in plastids (Troncoso-Ponce et al. 2011).
Author contribution statement  YX did the experiment and
manuscript writing. WX did the informatics analysis and
manuscript writing. ASM critically revised the manuscript.
ZC participated in the data analysis and manuscript revision.
HF and DH participated in the design of the study. BZ and
JZ did the gene expression validation experiments. ZM and
MP participated in manuscript revision. All authors read the
final manuscript and have given final approval of the version
to be published.
Acknowledgements  Thanks to Tingting Luo at Huazhong Agricul-
tural University for the technical help in fatty acid extraction. This
work was supported by grants from Hainan Natural Science Foun-
dation (No. 313058) and the Fundamental Scientific Research Funds
for Chinese Academy of Tropical Agricultural Sciences (Project No.
1630152018007, No. 1630152017004 and No. 1630152017005). ASM
is supported by DFG Emmy Noether grant MA6473/1-1.
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