Isolation and functional analysis of fatty acid desaturase genes from peanut (Arachis hypogaea L.)
Isolation and functional analysis of fatty acid desaturase genes from peanut (Arachis hypogaea L.)
Xiaoyuan Chi 0 1
Zhimeng Zhang 0 1
Na Chen 0 1
Xiaowen Zhang 1
Mian Wang 0 1
Mingna Chen 0 1
Tong Wang 0 1
Lijuan Pan 0 1
Jing Chen 0 1
Zhen Yang 0 1
Xiangyu Guan 1
Shanlin Yu 0 1
0 Shandong Peanut Research Institute , Qingdao, Shandong , P. R. China , 2 Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences , Qingdao, Shandong , P. R. China , 3 School of Ocean Sciences, China University of Geosciences , Beijing , P. R. China
1 Editor: Hong Zhang, Texas Tech University , UNITED STATES
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains.
Extensive studies of fatty acid desaturases have been done in many plants. However, less is known about the diversity of this gene family in peanut (Arachis hypogaea L.), an important oilseed crop that is cultivated worldwide.
In this study, twelve novel AhFADs genes were identified and isolated from peanut. Quanti
tative real-time PCR analysis indicated that the transcript abundances of AhFAB2-2 and
AhFAD3-1 were higher in seeds than in other tissues examined, whereas the AhADS and
AhFAD7-1 transcripts were more abundant in leaves. AhFAB2-3, AhFAD3-2, AhFAD4,
AhSLD-4, and AhDES genes were highly expressed in flowers, whereas AhFAD7-2,
AhSLD-2, and AhSLD-3 were expressed most strongly in stems. During seed development,
the expressions of AhFAB2-2, AhFAD3-1, AhFAD7-1, and AhSLD-3 gradually increased in
abundance, reached a maximum expression level, and then decreased. The AhFAB2-3,
AhFAD3-2, AhFAD4, AhADS, and AhDES transcript levels remained relatively high at the
initial stage of seed development, but decreased thereafter. The AhSLD-4 transcript level
remained relatively low at the initial stage of seed development, but showed a dramatic
increase in abundance at the final stage. The AhFAD7-2 and AhSLD-2 transcript levels
remained relatively high at the initial stage of seed development, but then decreased, and
finally increased again. The AhFAD transcripts were differentially expressed following
exposure to abiotic stresses or abscisic acid. Moreover, the functions of one AhFAD6 and four
AhSLD genes were confirmed by heterologous expression in Synechococcus elongates or
Shandong Province (ZR2014YL011 to MC and
ZR2014YL012 to XC), the Youth Scientific
Research Foundation of Shandong Academy of
Agricultural Sciences (2016YQN14 to NC), Qingdao
Civil Science and Technology Project
(14-2-3-34nsh to SY), Agricultural Scientific and
Technological Innovation Project of Shandong
Academy of Agricultural Sciences (CXGC2016B02
to JC), Young Talents Training Program of
Shandong Academy of Agricultural Sciences (to
XC). The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
Abbreviations: ADS, Δ9 desaturase; DES,
sphingolipid Δ4 desaturase; DesA, cyanobacterial
Δ12 desaturase; DesB, cyanobacterial ω3
desaturase; DesC, cyanobacterial Δ9 desaturase;
FAB2, stearoyl-ACP desaturase; FAD2, microsomal
Δ12 desaturase; FAD3, microsomal ω3 desaturase;
FAD4, trans Δ3 desaturase; FAD5, Δ7 desaturase;
FAD6, plastidial Δ12 desaturase; FAD7, plastidial
ω3 desaturase; FAD8, plastidial ω3 desaturase;
MGDG, monogalactosyldiacylglycerol; PG,
phosphatidylglycerol; SLD, sphingolipid Δ8
The present study provides valuable information that improves understanding of the biological roles of FAD genes in fatty acid synthesis, and will help peanut breeders improve the quality of peanut oil via molecular design breeding.
Peanut (Arachis hypogaea L.) is an allotetraploid species (2n = 4x = 40, AABB) cultivated
widely in tropical, subtropical and temperate regions [
]. The peanut seed is comprised of
around 50% oil, of which approximately 80% is oleic (36±67%) and linoleic (15±43%) acids
]. A high O/L ratio (ratio of oleic and linoleic acid) is the most desired oil quality trait as it
increases shelf life and the health benefits to manufacturers and consumers, respectively [
Improvements in peanut oil content and quality traits (high oleic and low linoleic acid) could
be accelerated by investigating the fatty acid biosynthesis pathway.
Fatty acid desaturases are responsible for the insertion of double bonds into pre-formed
fatty acid chains, and play an essential role in fatty acid metabolism and the maintenance of
biological membranes in living organisms [
]. They belong to a large gene family that contains
conserved histidine regions. Histidine-rich boxes are thought to form a part of the diiron
center where oxygen activation and substrate oxidation occur [
For higher plants, most information on the function and specificity of fatty acid desaturases
has come from characterization of Arabidopsis mutants that were deficient in specific
desaturation activities [
]. The desaturase genes detected in Arabidopsis are divided into several
subfamilies: stearoyl-ACP desaturase (FAB2), microsomal Δ12 desaturase (FAD2), plastidial Δ12
desaturase (FAD6), microsomal ω3 desaturase (FAD3), plastidial ω3 desaturase (FAD7),
plastidial ω3 desaturase (FAD8), trans Δ3 desaturase (FAD4), Δ7 desaturase (FAD5), Δ9 desaturase
(ADS), sphingolipid Δ8 desaturase (SLD), and sphingolipid Δ4 desaturase (DES). FAB2 is the
only soluble desaturase that has been characterized to date, and it catalyzes the desaturation of
stearic acid (C18:0) to C18:1 in the acyl carrier protein (ACP)-bound form [
]. FAD2 and
FAD6 are ω6 desaturases that synthesize dienoic linoleic fatty acid (C18:2) from oleic acid
(C18:1) in the endoplasmic reticulum (ER) and plastids, respectively. FAD3, FAD7, and FAD8
are ω3 desaturases that synthesize linolenic (C18:3) from linoleic (C18:2) acid in the ER
(FAD3) and plastids (FAD7 and FAD8) [
]. FAD4 and FAD5 specifically produce C16:1
from C16:0 for PG and MGDG, respectively . ADS is a Δ9 acyl-lipid desaturase that
participates in desaturation at the Δ9 position of C16:0 in the ER [
]. SLD encodes a sphingolipid
Δ8 desaturase that leads to the accumulation of 8 (Z/E)-C18-phytosphingenine in the leaves
and roots of Arabidopsis plants [
]. DES encodes the sphingolipid Δ4 desaturase
responsible for the synthesis of Δ4-unsaturated LCBs, such as sphingosine and sphinga-4,8-dienine in
To date, several types of fatty acid desaturase genes have been cloned and characterized
from peanut, including AhFAB2-1, AhSLD-1, four microsomal AhFAD2 and two chloroplast
AhFAD6 genes [
]. Among them, FAD2 is the most well-studied fatty acid desaturase
gene. Two microsomal oleoyl-PC desaturase genes (AhFAD2-1A and AhFAD2-1B), each
having its origin in different diploid progenitor species, have been isolated from cultivated peanut
[19±21]. Reduction in transcript levels or inactivation of both genes is required to produce
high O/L genotypes [22±26]. Different types of DNA markers from these two genes have been
developed to facilitate marker-assisted selection for the high-oleate trait [27±30]. The functions
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of AhFAD2-2 and AhFAD6 were verified by heterologous expression in S. cerevisiae. Linoleic
acid (18:2), normally not present in wild-type yeast cells, was detected in transformants of
these two genes .
The cultivated peanut (Arachis hypogaea L.) is derived from two wild diploid species
Arachis duranensis (A genome) and Arachis ipaensis (B genome) [
]. Now the genome
sequences of Arachis duranensis and Arachis ipaensis were released. The availability of these
genomes will lead to further advances in knowledge of genetic changes since the very recent
polyploidization event that gave rise to cultivated peanut and to an expanding knowledge of
understudied areas of plant biology [
]. In this study, we isolated twelve novel desaturase
genes from cultivated peanut. The expression patterns of these genes were investigated in
different tissues and at different stages of seed development. The expression of FAD genes in
response to abiotic stress and abscisic acid (ABA) was also analyzed. Additionally, the
functions of AhFAD6 and AhSLDs were confirmed by heterologous expression in Synechococcus
elongatus (strain PCC 7942) or yeast (Saccharomyces cerevisiae). Our results indicated that
these two types of genes are strong candidates for modifying fatty acid biosynthesis in peanut.
Results and discussion
Isolation of FAD genes from peanut
In a previous study, four fatty acid desaturases were isolated from peanut. These were
AhFAB2-1, AhFAD2-2, AhFAD6, and AhSLD-1 [
]. In this study, twelve new genes that
probably encode FAD proteins were found using Bioedit software [
]. They were cloned and
designated as AhFAB2-2, AhFAB2-3, AhFAD3-1, AhFAD3-2, AhFAD4, AhADS, AhFAD7-1,
AhFAD7-2, AhDES, AhSLD-2, AhSLD-3, and AhSLD-4, according to the homologous genes
identified in Arabidopsis (Table 1). Among the twelve genes, six had complete open reading
frames in the peanut cDNA library and were cloned by conventional RT-PCR. However, six
genes were cloned using the rapid amplification of cDNA ends (RACE) method. The open
reading frames of these genes ranged from 924 bp to1371 bp in length, and encoded 307 to 456
amino acids (Table 1 and S1 Table). The sequence information for the twelve genes was
submitted to Genbank along with their identification numbers (Table 1).
A search using NCBI BLAST revealed that the FAD proteins have high sequence similarities
with FADs in Arabidopsis. AhFAB2-2 and AhFAB2-3 shared 64.7% identity, which was higher
than that with AhFAB2-1 and AtFAB2-1. AhFAD3-1 shared about 74% sequence identities
with AhFAD3-2A, AhFAD3-2B, and AhFAD3-2C. They also shared high sequence identities
of about 66% with Arabidopsis AtFAD3. AhFAD4A shared a high sequence identity of 99.6%
with AhFAD4B, and both of them shared 53%, 50%, and 48% similarity with AtFAD4-1,
AtFAD4-2, and AtFAD4-3, respectively. AhADS shared 62.7%, 44%, and 41% amino acid
sequence identities with AtFAD5, AtADS-1, AtADS-2, respectively. The AhFAD7-2 protein
was most similar to AhFAD7-1A (76.8%) and AhFAD7-1B (76.8%). AhFAD7-1A,
AhFAD71B, and AhFAD7-2 showed more than 66% identity with AtFAD7 and AtFAD8. AhDES
showed 76% sequence similarity with AtDES. AhSLD-2 shares 61.2%, 94.8%, and 61.3%
amino acid sequence identities with AhSLD-1, AhSLD-3, and AhSLD-4, respectively.
AhSLD1, AhSLD-2, AhSLD-3, and AhSLD-4 showed 60.3%, 72.2%, 72.2%, and 61.7% identity,
respectively, with AtSLD-1, and 59.8%, 73.1%, 72.4%, and 61.9% identity to AtSLD-2, respectively.
Prediction of subcellular location by two programs, TargetP Server and Predotar, suggested
that the AhFAD7-1A, AhFAD7-1B, and FAD7-2 proteins were probably located in the
chloroplast, which is the same as AhFAB2-1 and AhFAD6. The first 73 amino acids at the N-terminal
end of the deduced proteins for these three genes had a high proportion of hydroxylated and
small, hydrophobic amino acids, which is typical of chloroplast transit peptides.
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The twelve desaturase genes contained typical histidine-rich boxes (S1±S3 Figs), which was
in accordance with the standard for different types of desaturase genes. For example, the two
histidine-boxes of AhFAB2-1, AhFAB2-2, and AhFAB2-3 genes were consistent with those of
plastidial stearoyl-ACP desaturases, which are represented as EENRHG and DEKRHE (S1
Fig). The three histidine-boxes of four microsomal Δ15 fatty acid desaturases (AhFAD3-1,
AhFAD3-2A, AhFAD3-2B, and AhFAD3-2C) and three plastidial Δ15 fatty acid desaturases
(AhFAD7-1A, AhFAD7-1B, and AhFAD7-2) genes matched the standards for Δ15 desaturase,
i.e. LGHDCGH, HR(K)THH, and HVIHH. The third histidine box of AhSLD-1, AhSLD-2,
AhSLD-3, and AhSLD-4 contained a His to Gln substitution at the third histidine residue,
which is also found in several fatty acid desaturases, such as the plant and animal fatty acid
Δ5and Δ6-desaturases [
]. Furthermore, in common with other desaturases of this type, four
AhSLD genes encoded proteins with a cytochrome b5-like hem-binding domain at the
N-terminus. The presence of this binding domain was characterized by the His-Pro-Gly-Gly motif,
which indicated that this putative desaturase existed as a fusion protein. Sphingolipid Δ4
desaturase activity has evolved independently of sphingolipid Δ8-desaturase activity . The
sphingolipid Δ4 desaturases shared only limited similarity with other proteins characterized by
the histidine box motifs (LAIHELSH, HLEHH, HNEHH), and they didn't contain a
cytochrome b5 domain (S2 Fig).
FAD4 encodes a predicted integral membrane protein that appears to be unrelated to classic
membrane bound fatty acid desaturases, based on overall sequence conservation. Its inferred
primary sequence has little resemblance to that of known fatty acid desaturases beyond the
presence of histidine motifs and membrane-spanning domains. However, the FAD4 protein
contains two histidine motifs resembling those of fatty acid desaturases [
]. The AhFAD4A
4 / 28
and AhFAD4B protein sequences contained two histidine motifs, HAWAH and HSAHH (S3
Fig), whose sequence and spacing are reminiscent of, but not identical to, conserved motifs in
membrane-bound desaturases [
]. While AhFAD4A and AhFAD4B contained a third
histidine motif (QGHH; S3 Fig), its sequence diverged from the third histidine motif present in
membrane-bound desaturases. Although glutamine substituted for histidine in these motifs
], known membrane-bound desaturases typically have two-to-three amino acids between
the glutamine and the histidines [
]. Another difference is that in characterized
membranebound desaturases, two histidine motifs are located between the second and the third
membrane spanning domains, while the third is located at the C-terminus of the protein. In
AhFAD4A and AhFAD4B, only one proposed histidine motif is located between membrane
spanning domains II and III, while the other two are located at the C-terminus (S3 Fig). Based
on these sequence features, it seems likely that FAD4 is a metalloenzyme that evolved
independently from the characterized desaturases [
To examine the relationships among different sources of FAD genes, sequences from
representative eukaryotic species belonging to plant monocotyledons (Oryza sativa,
Brachypodium distachyon, Setaria italica, and Zea may), eudicots (Arabidopsis thaliana, Glycine max,
Arachis duranensis, and Arachis ipaensis), a fern (Selaginella moellendorfii), a moss
(Physcomitrella patens), and algae (Chlamydomonas reinhardtii, Volvox carteri, Ostreococcus
lucimarinus, Micromonas pusilla RCC1545, and Coccomyxa subellipsoidea C-169), were selected to
construct a phylogenetic tree by the neighbor-joining method (Figs 1±3). No homologues of
any ADS genes were found in the genomes of rice (Oryza sativa), Brachypodium distachyon,
Setaria italica, or maize (Zea mays), which suggested that ADS genes may be absent in
The polyunsaturated fatty acids are synthesized by two distinct pathways in plants, known
as the prokaryotic and eukaryotic pathways, which are located within the membrane of plastids
and the endoplasmic reticulum (ER), respectively [
]. Therefore, plant desaturases fall into
two major classes: soluble and membrane-bound desaturases. The soluble desaturases are
analyzed separately from membrane-bound desaturases because they are restricted to higher
plants and show no evolutionary relationship with the more widely distributed membrane
The plant stearoyl-ACP desaturase is the only soluble desaturase identified to date. In
contrast, all other desaturases identified in plants, algae, animals, and fungi are integral membrane
]. The phylogenetic tree indicated that three AhFAB2 genes were grouped with
the stearoyl-ACP desaturases of higher plants and were distinct from those of the fern, the
moss, and the green algae (Fig 1). This may suggest that stearoyl-ACP desaturases in the fern/
moss/green algae and higher plants arose by independent gene duplication events. The three
AhFAB2 genes were clustered into two subgroups. The AhFAB2-2 and AhFAB2-3 proteins
were grouped together with FAB2 enzymes from eudicots and were separate from those of
monocotyledons, while AhFAB2-1 and AtFAB2-1 clustered together with genes from eudicots
and were set apart from the monocotyledon genes.
FAD4 encodes a predicted integral membrane protein that evolved independently from
classic membrane bound desaturases, which were analyzed separately from the remaining
membrane-bound desaturases [
]. Moreover this protein class, originally designated Kua
], is highly conserved in organisms ranging from bacteria (but not cyanobacteria)
to mammals. Unfortunately, definitive functional data are not available for these proteins. The
phylogenetic tree showed that the AhFAD4 protein was more closely related to enzymes from
5 / 28
Fig 1. Phylogenetic tree of stearoyl-ACP desaturase gene families reconstructed by the neighbor-joining (NJ) method. Gene sequences were
shown by their strain names, accession numbers (locus tags), or labels. Colored branches indicated different groups of proteins. Red: eudicot, blue:
monocotyledon, purple: fern, moss and algae. Bootstrapping with 1,000 replicates was used to establish the confidence limits of the tree branches.
6 / 28
Fig 2. Phylogenetic tree of trans Δ3 desaturase gene families reconstructed by the neighbor-joining
(NJ) method. Gene sequences were shown by their strain names, accession numbers (locus tags), or labels.
Bootstrapping with 1,000 replicates was used to establish the confidence limits of the tree branches.
higher plants, the fern, the moss, and the green algae, and were separate from those of bacteria
and animals (Fig 2).
As shown in the phylogenetic tree (Fig 3), all of the remaining membrane-bound
desaturases fell into four distinct subfamilies: the Δ7/Δ9 desaturase subfamily, the Δ12/ω3
7 / 28
Fig 3. Phylogenetic tree of membrane desaturase gene families reconstructed by the neighbor-joining (NJ) method. Gene sequences were shown
by their strain names, accession numbers (locus tags), or labels. Bootstrapping with 1,000 replicates was used to establish the confidence limits of the tree
branches. Colored branches indicated different groups of proteins. Red: Δ15 desaturase, blue: Δ7/Δ9 desaturase, purple: sphingolipid Δ4 desaturase,
green: microsomal Δ12 desaturase, orange: plastidial Δ12 desaturase, pink: sphingolipid Δ8 desaturase.
8 / 28
desaturase subfamily, the sphingolipid Δ8 desaturase subfamily, and the sphingolipid Δ4
desaturase subfamily. Δ9 desaturase is assumed to be the ancestor of the remaining desaturases
based on functional criteria and the position of the clade integrated by Δ9 desaturases [
The AhADS gene was grouped with eudicot Δ7 homologs and was set apart from Δ7 enzymes
in the fern, the moss, and the green algae, while the ADS genes in green algae and
cyanobacteria were placed in a basal position.
In the Δ12/ω3 desaturase subfamily, the AhFAD6 gene, grouped to eudicot chloroplastic
Δ12 desaturase, was located along with the cyanobacterial Δ12 desaturases at the basal position
of the tree. The higher plant microsomal Δ12 desaturases formed a group and were set apart
from those of the fern and the green algae. The AhFAD2-1A and AhFAD2-1B genes clustered
together and were separate from the AhFAD2-2 gene. The cyanobacterial ω3 desaturases were
placed in a basal position and were grouped with both microsomal and chloroplastic ω3
desaturases from higher plants, the fern, the moss, and the green algae. Seven putative ω3
desaturases from peanut (four FAD3 and three FAD7) were grouped with their respective
microsomal or chloroplastic ω3 desaturases from higher plants and were separate from the
fern, moss, and green algae enzymes. Therefore, it can be speculated that the cyanobacterial
Δ12 desaturase might be the origin of plant Δ12 and ω3 desaturases, including both chloroplast
and ER isozymes.
The sphingolipid Δ8 desaturases formed a separate clade (Fig 3). The SLD genes of green
algae were placed in a basal position. The four peanut sphingolipid Δ8 desaturase genes
(AhSLD-1, AhSLD-2, AhSLD-3, and AhSLD-4) clustered with SLDs from eudicots and were
separate from those of monocotyledons. The sphingolipid Δ4 desaturases integrated into one
clade (Fig 3). The AhDES gene, grouped with the eudicot DES genes, was located along with
DES desaturases from green algae at the basal position of the tree.
Tissue-specific expression patterns
Quantitative real-time PCR (qRT-PCR) was used to confirm the expression patterns of the
seventeen FAD genes in different peanut tissues and at different stages of seed development. The
actin 11 (AhACT11) gene was used as an internal reference control for total RNA input [
Figs 4 and 5 shows that these genes displayed specific temporal and spatial expression patterns
across different tissues and developmental stages. AhFAB2-2 and AhFAD3-1 transcripts were
more abundant in seeds than in any of the other tissues tested. The highest transcript
accumulations of AhFAB2-3, AhFAD3-2, AhFAD4, AhSLD-4, and AhDES genes occurred in flowers.
Among them, AhFAB2-3 and AhFAD3-2 had relatively higher expressions in stems. AhSLD-4
was expressed most strongly in flowers followed by seeds, whereas the expressions of the
AhFAD4 and AhDES gene were largely restricted to flowers. AhADS and AhFAD7-1 had
similar expression patterns, and were most abundant in leaves followed by flowers. The highest
abundances of AhFAD7-2 and AhSLD-2 transcripts were in stems. AhSLD-3 transcript levels
were highest in stems, followed by flowers, leaves, and roots, with the lowest levels being found
The expression patterns of FAD genes across six seed developmental stages are shown in
Fig 5. The AhSLD-3 transcript levels remained relatively low during the initial stage of seed
development, but increased gradually during the later stages, peaking at 40 DAP, and
decreased thereafter until 60 DAP. The expressions of AhFAB2-2 and AhFAD7-1 gradually
increased in abundance during seed development, reached a maximum expression level at 30
DAP, and then decreased thereafter. The AhFAB2-3, AhFAD3-2, and AhADS transcript levels
were highest at 10 DAP and decreased dramatically thereafter. The AhSLD-2 transcript level
remained relatively high at the initial stage of seed development, but decreased gradually from
9 / 28
Fig 4. Expression analysis of fatty acid desaturase genes of peanut using qRT-PCR in five peanut tissues. R, root; SM, stem; L, leaf; F, flower; SD,
seed. The relative mRNA abundance was normalized with respect to the peanut AhTUA5 gene. The bars were standard deviations (SD) of three biological
10 to 20 DAP, and then increased from 30 DAP. The AhFAD3-1 gene showed higher
expression levels at 20 DAPs and much lower levels during the other stages. The expression levels of
AhDES were highest at the initial stage of seed development, but gradually decreased in
abundance during the later stages. The AhSLD-4 transcript levels remained relatively low at the
initial stage of seed development, but showed a dramatic increase in abundance at 60 DAP. The
AhFAD4 transcript levels remained relatively high in the initial five stages, but showed a
Fig 5. Expression analysis of fatty acid desaturase genes of peanut using qRT-PCR at six stages of seed development. DS (10 to 60 DAP): six
developmental stages of seeds. The relative mRNA abundance was normalized with respect to the peanut AhTUA5 gene. The bars were standard
deviations (SD) of three biological repeats.
10 / 28
dramatic decrease thereafter. The AhFAD7-2 transcript level remained relatively high at the
initial stage of seed development, decreased gradually from 10 to 30 DAP, and then increased
from 40 DAP, with the highest expression being seen at 60 DAP.
Many desaturase genes from different plant species have been studied and their expression
levels are regulated in a tissue-specific manner. Our results indicated that the expression of the
AhDES gene was largely restricted to flowers, which highly resembled the expression pattern of
its closest ortholog AtDES in Arabidopsis with preferential expressions in pollen and floral
]. It has been reported that AtFAD2 (AT3G12120) and AtFAD6 (AT4G30950) mRNAs
are present in various Arabidopsis tissues, including the roots, rosette leaves, cauline leaves,
stems, flowers, and siliques [
]. The AhFAD2-1, AhFAD2-2, and AhFAD6 genes were
expressed in all tissues surveyed, which was consistent with the Arabidopsis orthologs . In
Arabidopsis, AtSLD-1 (At3g61580) was ubiquitously expressed in all organs and most highly
expressed in the flowers. However, AtSLD-2 (At2g46210) was expressed in flowers and siliques,
but shows only low levels of expression in the leaves, stems, and roots [
]. The AhSLD-4 gene
transcript accumulated to the greatest extent in flowers, which was consistent with the
Arabidopsis orthologs. The AhSLD-1 transcript was more abundant in leaves, whereas AhSLD-2 and
AhSLD-3 were most strongly expressed in stems. In Arabidopsis, AtFAB2-1 (At2g43710) and
AtFAB2-7 (At5g16240) were expressed at high levels in the flower, stem and leaf tissues, but at
low levels in roots and siliques. AtFAB2-4 (At3g02620) was relatively highly expressed in roots
compared to its levels in the leaf, stem, flower and silique tissues, while AtFAB2-6 (At5g16230)
was highly expressed in the leaves, but not in the roots. The AtFAB2-5 (At3g02630) isoform
was expressed at high levels in the leaf, stem and flower tissues, but at low levels in roots and
siliques. The AtFAB2-3 (At3g02610) isoform was only expressed at low levels in the roots and
flowers, while AtFAB2-2 (At1g43800) was not detected in any of the tissues analyzed [
peanut, AhFAB2-1 and AhFAB2-2 showed higher transcript abundances in seeds than in any
of the other tissues tested [
], whereas the AhFAB2-3 gene accumulated in flowers. In
Arabidopsis, the AtADS-1 (At1g06080) gene was expressed most strongly in inflorescence meristems
followed by leaves and flowers, and very weakly in roots and seedpods. The AtADS-2
(At2g31360) gene was expressed strongly in all the analyzed organs, although the expression
level was higher in flowers and roots than in other organs [
]. In peanut, AhADS
transcript levels were higher in the leaves, followed by flowers, and the lowest levels occurred in
the seeds and roots. The AtFAD3 gene was expressed in both the leaves and roots, while the
AtFAD7 gene transcript was only observed in the photosynthetically active organs of
Arabidopsis . In peanut, the AhFAD3-1 transcript abundances were higher in seeds than in the other
tissues examined, whereas the AhFAD3-2 transcripts were more abundant in flowers. The
AhFAD7-1 gene transcript accumulation was highest in leaves, whereas AhFAD7-2 was
expressed most strongly in stems. In Gossypium raimondii, GrFAD3.2, GrFAD8.1, and GrSLD5
were expressed at high levels in roots. GrFAD2.3, GrSLD3, GrSLD2, GrSLD1, GrFAD3.1, and
GrDSD1 shared high expression levels in young stems. GrFAD5 and GrFAD6 displayed the
highest transcript abundance in cotyledons. And GrFAD7 and GrFAD2.4 were predominantly
expressed in leaves [
]. In Cucumber, three CsFAB2 genes were dominantly expressed in the
seedling leaves. For CsFAD2.1, CsFAD3 and CsFAD6, the highest expression levels were
detected in the leaves, whereas for CsFAD4, CsFAD5.1 and CsFAD7, the highest transcript
abundances were detected in the cotyledons. In the roots and hypocotyls, only trace expression
levels could be detected for any cucumber FAD gene except CsFAD2.1 and CsFAD3 [
Perilla frutescens, PfrFAD2 and PfrFAD3 genes were expressed in leaves and during all stages of
seed development, and their expression levels in 2- to 3-week-old developing seeds were
6.7and 25-fold higher than their expression in leaves, respectively. In contrast, although the
expression of PfrFAD7-1 and PfrFAD7-2 was similar to that of PfrFAD2 and PfrFAD3 genes in
11 / 28
leaves, their expressions were much lower in developing seeds [
]. Thus, the same type of
FAD genes from different plants may have different spatial expression patterns, which requires
Expression patterns of AhFADs in peanut under abiotic stress
We monitored changes to these transcripts in peanut leaves and roots to confirm the
expression patterns of these FAD genes, including five previously cloned desaturase genes, under
cold, salt, drought, and ABA stresses. Two microsomal oleoyl-PC desaturase genes
(AhFAD21A and AhFAD2-1B) have been identified in peanut and their open reading frames (ORFs)
were 99% identical [
]. Thus, the gene-specific primers used for the amplification of
AhFAD2-1 in our analysis recognized and amplified both AhFAD2-1A and AhFAD2-1B genes.
Fig 6 shows the expression patterns of these FAD genes in peanut leaves after cold treatment.
There were no obvious changes in the abundances of the AhFAB2-1, AhFAB2-2, AhFAB2-3,
AhFAD2-1, AhFAD2-2, AhFAD6, AhFAD3-1, AhFAD3-2, and AhDES transcripts in peanut
leaves after cold treatment. The expressions of AhFAD4, AhFAD7-2, and AhSLD-2 increased
slightly at 1 h after treatment, and then decreased from 3 h to 12 h. After 24 h, their transcript
levels reached a maximum, which were approximately 93-, 5- and 3-fold, higher than the
nontreated controls, respectively. The expression levels of AhADS increased under cold stress,
peaking at 6 h, and then decreased. The greatest increase was about 3.6-fold. The expression of
AhSLD-1 increased slightly after 1 h treatment with cold and then decreased. After 48 h,
expression of the AhSLD-1 transcript reached its maximum level, with a nearly 2-fold increase.
The transcript levels of AhFAD7-1, AhSLD-3, and AhSLD-4 gradually increased under cold
stress, peaking at 48 h, 24 h, and 48 h, with approximately 4-, 6- and 2-fold increases,
respectively, compared to the non-treated controls.
The expression patterns of AhFADs in peanut leaves and roots after treatment with 200
mM NaCl were also monitored (Fig 7 and S4 Fig). In leaves, there were no obvious changes in
the abundances of the AhFAB2-1, AhFAB2-2, AhFAD2-2, AhFAD3-1, AhFAD4, AhSLD-1,
AhSLD-4, and AhDES transcripts after salt treatment. The transcript levels of AhFAD6,
AhADS, AhFAD7-1, AhFAD7-2, and AhSLD-2 gradually increased under salt stress, peaking at
3 h, 1 h, 1 h, 3 h, and 3 h, respectively, and showed approximately 3-, 2-, 2-, 13-, and 3-fold
increases, respectively. The expressions of AhFAD2-1 and AhFAD3-2 gradually increased
under salt stress, with peak levels that were about 2- and 7-fold higher, respectively, at 48 h.
The expression of AhFAB2-3 increased slightly at 1 h after treatment, and then decreased from
3 h to 12 h. After 48 h, AhFAB2-3 transcripts reached a maximum level, with the greatest
increase being approximately 3-fold. The expression of AhSLD-3 slightly increased in the
leaves of seedlings subjected to salt stress, with about a 2-fold peak increase at 48 h.
In roots, the levels of the AhFAB2-1, AhFAD2-1, AhFAD2-2, AhFAD6, AhFAD3-1, and
AhSLD-1 transcripts increased, and reached maximum levels at 3 h after salt treatment, with
the greatest increases observed being about 2-, 2-, 3-, 3-, 13-, and 4-fold, respectively,
compared to the non-treated controls. The expressions of AhFAB2-2, AhFAD7-1, AhFAD7-2,
AhSLD-2, and AhSLD-3 increased under salt stress, with a peak level at 6 h in roots, where the
greatest increases were about 431-, 8-, 242-, 4-, and 9-fold, respectively, compared to the
nontreated controls. The expression levels of AhFAD3-2, AhSLD-4, AhFAD4, and AhDES increased
after salt treatment, peaking at 12 h or 24 h, with increases of approximately 48-, 4-, 23-, and
13-fold, respectively. The transcript levels of AhADS increased in roots under salt stress, with
peak expression levels that were 49-fold greater at 48 h compared to the non-treated controls.
The expression of AhFAB2-3 increased gradually from 1 to 12 h after salt treatment and then
12 / 28
Fig 6. Expression analysis of fatty acid desaturase genes of peanut using qRT-PCR in peanut leaves upon cold treatment. 0h to 72h, leaves
exposed to cold (4ÊC) treatment. The relative mRNA abundance was normalized with respect to the peanut AhTUA5 gene. The bars were standard
deviations (SD) of three biological repeats.
decreased. After 48 h, expression of the AhFAB2-3 transcript reached its maximum level, with
a near 4-fold increase compared to the non-treated controls.
A 20% solution of PEG-6000 was used to mimic drought stress to monitor the expression
patterns of AhFADs in peanut leaves and roots (Fig 8 and S4 Fig). There were no obvious
changes in the abundances of the AhFAD3-1, AhFAD7-1, AhSLD-1, AhADS, AhSLD-4, and
AhDES transcripts in peanut leaves after drought treatment. In leaves, the transcript levels of
AhFAB2-2, AhFAD6, AhFAD2-2, AhFAB2-1, AhFAD7-2, and AhFAD3-2 gradually increased
under salt stress, peaking at 1 h, 1 h, 3 h, 6 h, 6 h, and 24 h, respectively, with increases of
approximately 5-, 2-, 2-, 2-, 8-, and 3-fold, respectively, compared to the non-treated controls.
The expressions of AhFAB2-3, AhFAD2-1, and AhSLD-3 increased in the leaves of seedlings
subjected to drought stress, with peak level increases of about 2-, 3-, and 2-fold, respectively, at
72 h. The expression of AhFAD4 increased under drought stress, with a maximum increase of
about 31-fold observed at 6 h, and then decreased from 12 h to 48 h. At 72 h, the AhFAD4
transcript levels increased again. The expression of AhSLD-2 increased gradually from 1 to 12 h
after drought treatment and then decreased. After 72 h, expression of the AhSLD-2 transcript
reached a maximum level, with a nearly 2-fold increase compared to the non-treated controls.
13 / 28
Fig 7. Expression analysis of fatty acid desaturase genes of peanut using qRT-PCR in peanut leaves and roots under salt stress. 0h to 48h, leaves
exposed to high salt (200 mM NaCl) treatment. The relative mRNA abundance was normalized with respect to the peanut AhTUA5 gene. The bars were
standard deviations (SD) of three biological repeats.
In roots, there were no obvious changes in the abundances of the AhFAB2-3, AhFAD3-1,
and AhSLD-1 transcripts after drought treatment. The levels of AhFAD2-2, AhFAD6,
AhFAD32, AhFAD4, and AhFAD7-1 transcripts increased, and reached their maximum levels 6 h after
drought treatment, with the greatest increases observed being about 2-, 2-, 4-, 4-, and 26-fold
higher, respectively, than the non-treated controls. The transcript levels of AhFAD7-2,
AhSLD2, AhSLD-3, AhFAB2-2, AhDES, AhFAB2-1, and AhFAD2-1 gradually increased under salt
stress, peaking at 1 h, 1 h, 1 h, 3 h, 3 h, 12 h, and 24 h, with increases of approximately 19-, 21-,
14-, 231-, 9-, 4-, and 11-fold, respectively, compared to the non-treated controls. The
expression of AhADS gradually increased under drought stress, with a maximum increase of about
4-fold observed at 72 h. The expression of AhSLD-4 increased under drought stress, with a
maximum increase of about 43-fold observed at 3 h, and then decreased from 6 h to 12 h.
After 24 h, the AhSLD-4 transcript levels increased again.
We also examined the response of AhFAD genes to exogenously applied ABA, which is a
plant signaling molecule involved in plant defense signaling pathways (Fig 9 and S4 Fig). In
leaves, there were no obvious changes in the abundances of the AhFAB2-1, AhFAB2-3,
AhFAD2-1, AhFAD2-2, AhFAD3-1, AhFAD3-2, AhFAD6, AhADS, AhFAD7-1, AhSLD-1,
AhSLD-2, AhSLD-3, AhSLD-4, and AhDES transcripts after ABA treatment. The transcript
levels of AhFAD7-2, and AhFAB2-2 increased after ABA treatment, peaking at 1 h and 48 h,
respectively, with increases of approximately 40- and 2- fold compared to the non-treated
14 / 28
Fig 8. Expression analysis of fatty acid desaturase genes of peanut using qRT-PCR in peanut leaves and roots under drought stress. 0h to 72h,
leaves exposed to 20% PEG-6000 treatment. The relative mRNA abundance was normalized with respect to the peanut AhTUA5 gene. The bars were
standard deviations (SD) of three biological repeats.
controls. The expression of AhFAD4 increased rapidly 1 h after treatment, and then decreased
from 3 h to 12 h. After 24 h, AhFAD4 transcripts reached a maximum level, with an
approximately 2-fold increase.
In roots, there were no obvious changes in the abundances of the AhFAB2-3, AhFAD3-1,
AhADS, and AhSLD-4 transcripts after ABA treatment. The transcript levels of AhSLD-2,
AhSLD-3, AhFAD2-2, AhFAD6, AhSLD-1, AhFAD3-2, and AhDES gradually increased after
ABA treatment, peaking at 1 h, 1 h, 3 h, 3 h, 12 h, 24 h, and 24 h, respectively, with increases of
approximately 13-, 22-, 3-, 3-, 3-, 5-, and 5-fold compared to the non-treated controls. The
AhFAD2-1, AhFAD7-1, and AhFAD4 transcripts levels were considerably higher in
ABAtreated roots than in untreated roots after 72 h, with a maximum increase of approximately 5-,
3-, and 8-fold, respectively. The AhFAD7-2 and AhFAB2-1 expressions increased after 1 h or 3
h treatment with ABA and then decreased. After 24 h, the AhFAD7-2 and AhFAB2-1 transcript
levels reached a maximum, with the greatest increase being approximately 37- and 2-fold,
respectively. The AhFAB2-2 transcript levels increased after 6 h treatment, but decreased from
12 h to 24 h. After 48 h, the expression of AhFAB2-2 increased with a peak level of 757-fold.
Environmental factors compel organisms to acclimatize to the external conditions [
Poikilothermic organisms, such as cyanobacteria and plants, modulate the composition of
their membrane lipids in response to changes in environmental conditions [
15 / 28
Fig 9. Expression analysis of fatty acid desaturase genes of peanut using qRT-PCR in peanut leaves and roots under ABA treatment. 0h to 72h,
leaves exposed to 100uM ABA treatment. The relative mRNA abundance was normalized with respect to the peanut AhTUA5 gene. The bars were standard
deviations (SD) of three biological repeats.
Unsaturated fatty acids are essential constituents of glycerolipids in biological membranes and
the unsaturation level of membrane lipids is important in controlling the fluidity of
]. The extent of unsaturation is mainly determined by the activity of fatty acid
desaturases, the enzymes that introduce double bonds into specific positions of lipid fatty-acyl
]. Previous studies have revealed that FAD genes are crucial for the survival of plants
faced with different environmental stresses. In Arabidopsis, the expression of AtFAD8 was
strongly induced by cold temperatures [
]. AtFAD2 and AtFAD6 were found to be active in
seedlings under salinity stress [
]. The ads2 mutant Arabidopsis plants showed increased
sensitivity to chilling and freezing temperatures , and the mutants of AtSLD genes also
showed enhanced sensitivity to prolonged low-temperature exposure [
]. In transgenic
tobacco plants, over-expressing Arabidopsis AtFAD7 also enhanced cold tolerance [
whereas antisense expression of AtFAD7 reduced salt and drought tolerance [
plants overexpressing either Arabidopsis AtFAD3 or AtFAD8 gene also exhibited increased
tolerance to drought and osmotic stress [
]. In Saussurea involucrate, the expression of
sikSACPD increased in leaves as the temperature decreased from 20 to -10ÊC. The FAB2:
SikSACPD transgenic plants showed a slightly more resistance to the freezing stress than the
FAB2:FAB2 transgenic plants and the wild-type [
]. In Gossypium raimondii, GrFAD8.1,
GrFAD2.2, GrFAD8.2, GrSLD2, GrSLD4, GrDSD1 and GrSLD5 were found to be significantly
16 / 28
up-regulated in response to cold stress. Conversely, GrFAD5, GrFAD7, GrFAD2.3, GrSLD1
and GrSLD3 were heavily down-regulated after long periods of cold stress treatment [
safflower, the transcription level of CtFAD3 remained constant at all different growth
temperatures in the leaves; in contrast, the accumulation of CtFAD7 mRNA slightly increased at low
temperature, while CtFAD8 mRNA decreased significantly. The expressions of CtFAD3,
CtFAD7, and CtFAD8 in the roots significantly increased at low temperature [
]. In rice,
OsFAD8 has been reported to have a functional role in stress tolerance at low temperatures
]. In tomato, LeFAD3 overexpression enhanced the tolerance of tomato seedlings for
salinity stress [
], whereas silencing the LeFAD7 gene alleviated high-temperature stress [
soybean, the expression of FAD3 and FAD7 was tightly regulated in response to cold
Our results indicated that FAD transcripts from peanut were differentially expressed
following exposure to abiotic stresses or a stress-induced plant hormone. The AhFAD7-2 transcript
levels were considerably enhanced under all stress treatments. The expressions of AhFAD4,
AhSLD-2, and AhSLD-3 increased in all materials after the stress treatments, except for salt- or
ABA-treated leaves, whereas the transcript levels of AhFAD3-1 only increased in salt-stressed
roots. The AhFAB2-2, AhFAD2-1, AhFAD6, and AhFAD2-3 transcript levels were distinctly
enhanced after exposure to four kinds of stress separately, except for cold-, salt-treated leaves
or cold-, ABA-treated leaves. The expressions of AhFAD7-1 increased in all materials after the
stress treatments, except for drought- and ABA-treated leaves. The transcript levels of
AhFAB2-1 and AhFAD2-2 increased in salt-, drought-, and ABA-treated roots, and
droughttreated leaves, whereas the expression of AhADS increased in cold-, salt-treated leaves and
salt, drought-treated roots. The AhDES transcripts levels increased substantially in roots exposed
to salt, drought, and ABA stresses, whereas the AhSLD-4 transcript levels were distinctly
enhanced in salt-, drought-treated roots and cold-treated leaves. AhSLD-1 expression
increased in salt- or ABA-treated roots and cold-treated leaves, whereas AhFAB2-3 transcript
levels increased in salt-, drought-stressed leaves and salt-treated roots. Taken together, these
results from qRT-PCR suggested that the expression of most peanut FAD genes was induced
by stress treatment, consistent with the FAD genes from other plants [61±70]. To
comprehensively decipher their functions involved in stress tolerance in peanut seedlings, some lipidomic
and transcriptomic methods would be employed.
Heterologous expression of AhFAD6 in Synechococcus elongatus
(strain PCC 7942)
Synechococcus elongatus (strain PCC 7942) is a freshwater unicellular cyanobacterium that
only contains monounsaturated fatty acids. It is an excellent model system for studying fatty
acid metabolism. When the Δ12 fatty acid desaturase gene is introduced into the genome of S.
elongates, the resultant cells will produce considerable amounts of diunsaturated fatty acids.
Heterologous expression in S. elongates was used to confirm Δ12 regioselectivity and the
function of AhFAD6 genes. The pYFAD6 and empty vector (pSyn_1, control) were
transformed into the S. elongates. The total lipids of the transformants were analyzed using GC-MS.
The results showed a novel fatty acid peak from pYFAD6, which was absent in the control.
The novel fatty acid was designated as C18:2 by comparing the retention time to FAME
standard mixtures (Sigma). No C16:2 was detected, which indicated that AhFAD6 recognized only
one substrate (C18:1) in S. elongates (Table 2). The C18:2 percentage was 3.3% for pYFAD6
transformants. These data showed that the activity of the AhFAD6 protein was significantly
higher in the cyanobacterium than in yeast, where the percentage of C18:2 was 0.1% [
Yeast is known to be the model of choice for the functional characterization of microsomal
17 / 28
a Dashes indicated that the fatty acid was not detected.
FADs because it contains the short electron transport system required by these desaturases
(i.e., cytochrome b5 and NADH-cytochrome b5 reductase) [
]. Nevertheless, the high
desaturation level evident from Table 2 suggested that desaturases of plastidial origin, which
usually require ferredoxin and NADPH-ferredoxin reductase, were supplied to some extent with
reducing equivalents in yeast cells.
It is well known that the AhFAD2-1 gene plays a major role in the conversion of oleic to
linoleic acid in seed storage oils [
19, 20, 72
]. Two other genes, AhFAD2-2 and AhFAD6 have been
isolated by us [
]. They also contribute to the C18:2 pool, although a major portion of this
pool reflects contributions from AhFAD2-1 activity. This may indicated that a switch from
oleic acid to linoleic acid might involve more Δ12 desaturase genes and an intricate metabolic
network that regulates linoleic acid biosynthesis between the endoplasmic reticulum and the
chloroplast within peanut cells. The functional validation of these two novel members would
facilitate the further genetic manipulation of the peanut oil quality trait that is based on a high
O/L ratio [
Heterologous expression of AhSLDs in Saccharomyces cerevisiae
In order to elucidate whether AhSLD-1, AhSLD-2, AhSLD-3, and AhSLD-4 encode functional
Δ8 sphingolipid desaturases, these four genes were expressed in S. cerevisiae under the control
of the galactose-inducible GAL1 promoter. Reverse-phase high-performance liquid
chromatography (RP-HPLC) showed that when transformed with the empty vector (control), yeast
cells showed an LCB pattern that was identical to the wild-type pattern, which contained
mainly C18-phytosphinganine (t18:0) (Fig 10). In contrast, yeast transformants containing
AhSLD-1, AhSLD-2, AhSLD-3, and AhSLD-4 accumulated novel (Z)- and (E)-desaturated
sphingoid bases with productions of 17.63%, 53.5%, 23.4%, and 0.92%, respectively, in
addition to t18:0 (Fig 10 and Table 3). The ratio of the newly synthesized
8(Z)-C18-phytosphinganine (t18:18Z) and 8(E)-C18-phytosphinganine (t18:18E) was quite different in the four
transformants: the ratio for AhSLD-1 was 7.74, and those for AhSLD-2 and AhSLD-3 were 0.13.
AhSLD-4 only newly synthesized t18:18Z. These results indicated that AhSLD-1, AhSLD-2,
AhSLD-3, and AhSLD-4 all encoded functional Δ8 sphingolipid desaturases with diverse
biochemical functions. In a similar way, four BrSLD1 genes in Brassica rapa have been isolated,
which also catalyze different ratios of t18:18Z and t18:18E [
]. However, two different genes
encoding sphingolipid Δ8 desaturase were discovered in Arabidopsis and Helianthus annuus,
there have been no reports on different product ratios in these two species [
48, 74, 75
study will help further elucidate the key domains determining t18:18Z and t18:18E biosynthesis
using domain swapping between these similar enzymes.
In conclusion, twelve novel FAD-like genes from peanut were cloned, including two FAB2,
two FAD3, two FAD7, one FAD4, one ADS, one DES, and three SLD genes. The functions of
18 / 28
Fig 10. Formation of phytosphingenines in yeast cells by heterologous expression of sphingolipid desaturase from peanut. A: t18:0, used as the
standard. B: the predominating LCBs from S. cerevisiae cells (INVSc1) harbouring the empty vector pYES2 with t18:0 as the control. C-F: formation of
t18:18Z and t18:18E in yeast cells expressing pYES2-AhSLD-1/2/3/4. LCBs from yeast cells were converted into their fluorescent derivatives and analyzed by
Conversion rate (%)a
19 / 28
aConversion rate (%) = (t18:18Z + t18:18E)/(t18:18Z+t18:18E+t18:0)×100.
b Dashes indicated that the t18:18E was not detected.
one AhFAD6 and four AhSLD genes were verified by heterologous expression in S. elongates or
S. cerevisiae. Better understanding of this enzyme family will improve efforts to modify the
content and composition of seed oils or improve abiotic stress resistance in plants. The results
generated in our study provide new information that will increase our understanding of the
evolution, functional diversity and gene expression of fatty acid desaturases in plants and
opens the way to select candidate genes for functional validation studies in peanut.
Materials and methods
No specific permits were required for the described field studies. No specific permissions were
required for these locations and activities. The location is not privately-owned or protected in
any way and the field studies did not involve endangered or protected species.
Peanut plants (A. hypogaea L. cultivar Huayu 19) were grown in a growth chamber with a 16 h
light/8 h dark photoperiod at 26ÊC/22ÊC day/night temperatures. Leaves, stems and roots were
sampled from the seedlings at the trefoil leaf stage. Seeds were sampled at 10, 20, 30, 40, 50,
and 60 days after pegging (DAP). Flowers were collected when the seedlings were in the
flowering phase. For the cold treatment, seedlings in the soil at the trefoil leaf stage were kept at
4ÊC, and leaves were sampled separately either before cold treatment (0 h) or after continuous
exposure to 4ÊC for 1, 3, 6, 12, 24, 48, or 72 h. For stress treatments, roots of seedlings grown
in soil were flushed carefully with tap water to remove all soil, and then submerged in solutions
of 200 mM NaCl, 20% PEG-6000, or 100 μM ABA. Leaves and roots were sampled separately
after treatment for 0, 1, 3, 6, 12, 24, 48, or 72 h. All samples were immediately frozen in liquid
nitrogen and stored at -80ÊC until required.
Identification of FAD family genes in a peanut cDNA library using Bioedit software
The amino acid sequences of FAD genes of Arabidopsis, AtFAB2-1 (AT2G43710), AtADS-1
(AT1G06080), AtFAD2 (AT3G12120), AtFAD4-1 (AT4G27030), AtFAD6 (AT4G30950),
AtFAD7 (AT3G11170), AtFAD8 (AT5G05580), AtFAD3 (AT2G29980), AtFAD5
(AT3G15850), and AtDES (AT4G04930), were used as query to search for homologous genes
from the peanut cDNA library including 36,741 cDNA sequences. Before searching for
members of the FAD gene family, a local nucleotide database file was created using Bioedit software
]. A local BLAST procedure was then run to find the homologous genes of the FAD family.
Using this method, we found twelve genes that may encode FAD proteins.
Isolation of full-length cDNA sequences
Total RNA was extracted using the RNeasy Plant Mini kit (Qiagen, Valencia, CA, USA).
Contamination with genomic DNA was eliminated by treatment with recombinant DNase I
(Qiagen), as recommended by the vendor. Only RNA preparations having an A260/A280 ratio of
1.8±2.0 and an A260/A230 ratio >2.0 were used for subsequent analysis. The integrity of RNA
was verified by electrophoresis through 2% agarose gels, followed by SYBR Green staining.
First-strand cDNA synthesis was carried out with 2 μg RNA using an RT-PCR kit (Promega,
Madison, WI, USA) according to the manufacturer's procedure.
We performed PCR with the LA PCR system (Takara, Dalian, China), using 2.5 μl of
10×PCR buffer with MgCl2, 1 μl of each primer (10 μM) (S2 Table), 4.0 μl of 10 mM dNTPs,
20 / 28
1 μl of cDNA sample, 0.5 μl of LA Taq™ DNA polymerase, and 15 μl of double-distilled water.
The PCR products were separated by electrophoresis through a 1% agarose gel, and purified
using a Gel Extraction Kit (Takara) according to the manufacturer's protocol. The purified
products were then cloned into the pMD18-T Easy vector (Takara) and sequenced (Shangon,
The open reading frames (ORFs) and encoded amino acid sequences of all genes were deduced
using BioXM 2.6. Physicochemical properties of the deduced proteins were predicted using
Protparam (http://www.expasy.ch/tools/protparam.html). Active sites of the protein sequences
were analyzed by comparisons against the PROSITE database. The putative subcellular
localizations of the candidate proteins were estimated by TargetP (http://www.cbs.dtu.dk/services/
TargetP/) and Predotar (http://urgi.versailles.inra.fr/predotar/predotar.html).
Homologs of each member of the Arabidopsis FAD family were identified by BLASTP searches
with datasets from Phytozome v10.3 (www.phytozome.net) and Peanut Genome Project
]. Only those sequences with an e-value less than e−50 were
considered as members of the FAD family. In each tree, gene sequences were displayed using
the nomenclature with the following abbreviations: At, Arabidopsis thaliana TAIR10; Glyma,
Glycine max Wm82.a2.v1; Ah, Arachis hypogaea L.; Ai, Arachis ipaensis; Ad, Arachis
duranensis; LOC_Os, Oryza sativa v7.0; Bradi, Brachypodium distachyon v2.1; Si, Setaria italic v2.1;
GRMZM, Zea may 6a; Phpat, Physcomitrella patens v3.0; Sm, Selaginella moellendorffii v1.0;
Cre, Chlamydomonas reinhardtii v5.5; Vocar, Volvox carteri v2.0; Ol, Ostreococcus lucimarinus
V2.0; Mp, Micromonas pusilla RCC1545 v3.0; Cs, Coccomyxa subellipsoidea C-169 v2.0. The
other amino acid sequences beyond the 16 species were retrieved from NCBI (http://www.
ncbi.nlm.nih.gov/). S3 Table provides a detailed description of the proteins used and the
corresponding accession numbers. S4 Table provides the pairwise comparison of the FADs from
cultivated and wild peanut varieties. Amino acid sequences were aligned using the ClustalX
program with the implanted BioEdit [
]. The neighbor-joining (NJ) method in MEGA4 [
was used to construct the phylogenetic tree. Bootstrapping with 1,000 replicates was used to
establish the confidence limits of the tree branches. Default program parameters were used.
Quantitative real-time RT-PCR
qRT-PCR analysis was performed using a LightCycler 2.0 instrument system (Roche,
Germany). The actin 11 gene (AhACT11) was selected as the reference gene [
]. Seventeen pairs
of gene-specific primers (S5 Table) were designed after analyses of the target genes' sequences.
qRT-PCR reactions were performed using the SYBR Premix Ex Taq polymerase (Takara)
according to the manufacturer's instructions. Each 20-μl reaction was comprised of 2 μl of
template, 10 μl of 2× SYBR Premix, and 0.4 μl (200 nM) of each primer. The reactions were
subjected to an initial denaturation step of 95ÊC/10 s, followed by 40 cycles of 95ÊC/5 s, 60ÊC/
30 s and 72ÊC/10 s. A melting curve analysis was performed at the end of the PCR run over the
60±95ÊC range, increasing the temperature stepwise by 0.5ÊC every 10 s. The baseline and
quantification cycle (CP) were automatically determined using the LightCycler Software. Zero
template controls were included for each primer pair, and each PCR reaction was carried out
in triplicate. The relative quantification method (delta-delta Cp) was used to evaluate
quantitative variation [
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Heterologous expression of AhFAD6 in Synechococcus elongatus
(strain PCC 7942)
The AhFAD6 in the pSyn_1 plasmid was transformed into the freshwater unicellular
cyanobacterium S. elongatus (strain PCC 7942), using the natural transformation method according
to the manual (Invitrogen, Carlsbad, CA, USA). Transformants were selected by screening for
resistance to 10 μg/mL spectinomycin in BG-11 solid medium. Colony PCR was performed to
screen the transformed S. elongatus colonies for full integration of the promoter and the gene
of interest. The positive colonies were transferred into BG-11 liquid medium with 10 μg/mL
spectinomycin and grown at 28ÊC for 30 days. Cells were harvested by centrifugation, washed
three times with double-distilled water and used for the extraction of total fatty acids.
Fatty acid extraction and analysis
Total fatty acids were extracted and transmethylated with methanolic HCl from algae cells
according to Browse et al (1986) [
]. All samples were analyzed using a 7890A/5975C gas
chromatography (Agilent Technologies, California, USA) equipped with a 5975C single
quadrupole GC/MSD detector and an HP-INNOWAX capillary column (30
m × 250 μm × 0.25 μm). High purity nitrogen was used as the carrier gas with flow rate of 40
mL/min. The injector and detector temperatures were both 250ÊC, and the column
temperatures were programmed from 150ÊC to 230ÊC. Measurements were performed using peak
height area integrals expressed as a percentage of the total of all integrals. The experiment was
carried out in triplicate.
Heterologous expression of AhSLDs in Saccharomyces cerevisiae
The AhSLDs in the pYES2 plasmid were transformed into the auxotrophic S. cerevisiae strain
INVSc1 (MATa his3-Δ1 leu2 trp1-289 ura3-52), using the polyethylene glycol/lithium acetate
method according to the manual (Invitrogen, Carlsbad, CA, USA). Yeast cells transformed
with an empty pYES2 plasmid were used as the negative control. The AhSLDs-transformed
yeasts were grown at 30ÊC in SC-U containing 2% (w/v) glucose for 24 h, and expression was
further induced by the addition of 2% (w/v) galactose and 1% (w/v) tergitol NP-40 (Sigma,
Taufkirchen, Germany) for an additional 72 h at 20ÊC. Yeast cells were collected by
centrifugation for 10 min at 2000 g and dried at 50ÊC.
Long chain base (LCB) analyses
Pellets of wild-type and transformed yeast cells (100 mg of dried weight) were used to prepare
the LCBs for subsequent RP-HPLC analysis as previously described [
(t18:0) (Sigma) was used as the internal standard for yeast sample analyses. Briefly, induced
yeast cells (100 mg, dried weight) were grounded into a fine powder, and subjected to strong
alkaline hydrolysis in 2 ml of 10% (w/v) aqueous Ba(OH)2 and 2 ml of dioxane for 16 h at
110ÊC. After hydrolysis, 2 ml of 2% (w/v) ammonium sulphate was added, and the liberated
sphingolipid long chain bases were extracted with 2 ml of diethylether. The upper phase was
removed to a second tube, dried under nitrogen, and derivatized with o-phthaldialdehyde
(OPA) (Invitrogen, Carlsbad, CA, USA) as previously described [
]. Individual LCBs were
separated by RP-HPLC (Waters alliance 2695±2475 multi λ fluorescence detector, Waters,
Milford, USA) with Penomenex C18 columns (250 mm 4.6 mm, 5 mm). Elution was
performed at 1.2 mL/min with 20% solvent RA (5 mmol/L potassium phosphate, pH 7.0), 80%
solvent RB (100% methanol) for 9 min, increasing to 90% solvent RB by 32 min, returning to
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80% solvent RB and re-equilibrating for 2 min. Fluorescence was excited at 340 nm and
detected at 455 nm.
S1 Fig. Alignment of deduced amino acid sequences of stearoyl-ACP desaturase genes of
peanut and Arabidopsis. Identical amino acid residues were highlighted in black. The
conserved histidine motifs were highlighted in black boxes.
S2 Fig. Alignment of the deduced amino acid sequences of membrane desaturase genes.
Identical amino acid residues were highlighted in black. The conserved histidine motifs were
highlighted in black boxes.
S3 Fig. Alignment of the deduced amino acid sequences of trans Δ3 desaturase genes.
Identical amino acid residues were highlighted in black. The conserved histidine motifs were
highlighted in black boxes.
S4 Fig. Expression analysis of several fatty acid desaturase genes of peanut using qRT-PCR
in peanut leaves under salt, drought or ABA treatment.
S1 Table. Difference of closely related multiple AhFADs.
S2 Table. DNA sequences of oligonucleotide primers used for gene cloning and vector
construction in this study.
S3 Table. The fatty acid desaturase enzyme homologs used for the phylogenetic analyses.
The table shows the species, gene names and accession numbers of the sequences used in the
S4 Table. Pairwise comparison of the FADs from cultivated and wild peanut varieties.
S5 Table. DNA sequences of oligonucleotide primers used for qRT-PCR in this study.
Data curation: Xiaowen Zhang.
Formal analysis: Na Chen.
Investigation: Xiaoyuan Chi.
Methodology: Tong Wang, Jing Chen.
Funding acquisition: Xiaoyuan Chi, Zhimeng Zhang, Na Chen, Mingna Chen, Tong Wang,
Jing Chen, Shanlin Yu.
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Project administration: Shanlin Yu.
Resources: Zhimeng Zhang.
Software: Mingna Chen, Zhen Yang.
Supervision: Xiangyu Guan, Shanlin Yu.
Validation: Lijuan Pan.
Visualization: Mian Wang.
Writing ± original draft: Xiaoyuan Chi.
Writing ± review & editing: Xiaoyuan Chi.
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1. Chi XY , Hu RB , Zhang XW , Chen MN , Chen N , Pan LJ , et al. Cloning and functional analysis of three diacylglycerol acyltransferase genes from peanut (Arachis hypogaea L.) . PLoS ONE . 2014 ; 9 ( 9 ): e105834. https://doi.org/10.1371/journal.pone. 0105834 PMID: 25181516
2. Moore KM , Knauft DA . The inheritance of high±oleic acid in peanut . J Hered . 1989 ; 80 : 252 ± 3 .
3. Pandey MP , Wang ML , Qiao LX , Feng SP , Khera P , Wang H , et al. Identification of QTLs associated with oil content and mapping FAD2 genes and their relative contribution to oil quality in peanut (Arachis hypogaea L.) . BMC Genetics . 2014 ; 15 : 133 . https://doi.org/10.1186/s12863-014 -0133-4 PMID: 25491595
4. Singh SC , Sinha RP , HaÈder DP . Role of lipids and fatty acids in stress tolerance in cyanobacteria . Acta Protozoo . 2002 ; 41 : 297 ± 308 .
5. Chi XY , Yang QL , Zhao FQ , Qin S , Yang Y , Shen JJ , et al. Comparative analysis of fatty acid desaturases in cyanobacterial genomes . Comp Funct Genom. 2008a; 2008 : 1 ± 25 .
6. Chi XY , Zhang XW , Guan XY , Ding L , Li YX , Wang MQ , Qin S. Fatty acid biosynthesis in eukaryotic photosynthetic microalgae: identification of a microsomal delta 12 desaturase in Chlamydomonas reinhardtii . The Journal of Microbiology. 2008b; 46 : 189 ± 201 .
7. Somerville C , Browse J . Plant lipids: metabolism mutants and membranes . Science . 1991 ; 252 : 80 ±7. https://doi.org/10.1126/science.252.5002.80 PMID: 17739077
8. Murphy DJ , Piffanelli P . Fatty acid desaturases: structure, mechanism and regulation . In: Harwood HL, ed. Plant lipid biosynthesis . Cambridge: Cambridge University Press; 1998 . p. 95 ± 130 .
9. Gibson S , Arondel V , Iba K , Somerville C . Cloning of a temperature-regulated gene encoding a chloroplast ω-3 desaturase from Arabidopsis thaliana . Plant Physiology . 1994 ; 106 : 1615 ± 21 . PMID: 7846164
10. Berberich T , Harada M , Sugawara K , Kodama H , Iba K , Kusano T . Two maize genes encoding ω-3 fatty acid desaturase and their differential expression to temperature . Plant Molecular Biology . 1998 ; 36 : 297 ± 306 . PMID: 9484441
11. Fukuchi±Mizutani M , Tasaka Y , Tanaka Y , Ashikari T , Kusumi T , Murata N. Characterization of Δ9 acyllipid desaturase homologues from Arabidopsis thaliana . Plant Cell Physiol . 1998 ; 39 ( 2 ): 247 ± 53 . PMID: 9559566
12. Heilmann I , Pidkowich MS , Girke T , Shanklin J . Switching desaturase enzyme specificity by alternate subcellular targeting . PNAS . 2004 ; 101 ( 28 ): 10266 ± 71 . https://doi.org/10.1073/pnas.0402200101 PMID: 15240892
13. Sperling P , Zahringer U , Heinz E. A sphingolipid desaturase from higher plants. Identification of a new cytochrome b5 fusion protein . J Biol Chem . 1998 ; 273 : 28590 ± 6 . PMID: 9786850
14. Ryan PR , Liu Q , Sperling P , Dong B , Franke S , Delhaize E. A higher plant Δ8 sphingolipid desaturase with a preference for (z)-isomer formation confers aluminum tolerance to yeast and plants . Plant Physiology . 2007 ; 144 : 1968 ±77. https://doi.org/10.1104/pp. 107 .100446 PMID: 17600137
15. Ternes P , Franke S , ZaÈhringer U , Sperling P , Heinz E . Identification and characterization of a sphingolipid Δ4-desaturase family . Journal of Biological Chemistry . 2002 ; 77 ( 28 ): 25512 ± 8 .
16. Michaelson LV , ZaÈuner S , Markham JE , Haslam RP , Desikan R , Mugford S , et al. Functional characterization of a higher plant sphingolipid Δ4-desaturase: defining the role of sphingosine and sphingosine± 1±phosphate in Arabidopsis . Plant Physiology . 2009 ; 149 : 487 ± 98 . https://doi.org/10.1104/pp. 108 . 129411 PMID: 18978071
17. Chi XY , Yang QL , Pan LJ , Chen MN , He YN , Yang Z , et al. Isolation and characterization of fatty acid desaturase genes from peanut (Arachis hypogaea L.) . Plant Cell Rep . 2011 ; 30 : 1393 ± 404 . https://doi. org/10.1007/s00299-011 -1048-4 PMID: 21409552
18. Wang Y , Zhang X , Zhao Y , Prakash CS , He G , Yin D. Insights into the novel members of the FAD2 gene family involved in high-oleate fluxes in peanut . Genome . 2015 ; 58 ( 8 ): 375 ± 83 . https://doi.org/10. 1139/gen-2015 -0008 PMID: 26332746
19. Jung S , Powell G , Moore K , Abbott A. The high oleate trait in the cultivated peanut (Arachis hypogaea L.) . II. Molecular basis and genetics of the trait . Mol Gen Genet . 2000a ; 263 : 806 ± 11 . PMID: 10905348
20. Jung S , Swift D , Sengoku E , Patel M , TeuleÂ F , Powell G , et al. The high oleate trait in the cultivated peanut (Arachis hypogaea L.). I. Isolation and characterization of two genes encoding microsomal oleoylPC desaturases . Mol Gen Genet . 2000b ; 263 : 796 ± 805 .
21. Lopez Y , Nadaf HL , Smith OD , Connell JP , Reddy AS , Fritz AK . Isolation and characterization of the delta(12)-fatty acid desaturase in peanut (Arachis hypogaea L.) and search for polymorphisms for the high oleate trait in Spanish market-type lines . Theor Appl Genet . 2000 ; 101 : 1131 ± 38 .
22. Chu Y , Ramos L , Holbrook CC , Ozias±Akins P. Two alleles of AhFAD2B control the high oleic acid trait in cultivated peanut . Crop Sci . 2009 ; 49 : 2029 ± 36 .
23. Patel M , Jung S , Moore K , Powell G , Ainsworth C , Abbott A. High-oleate peanut mutants result from a MITE insertion into the FAD2 gene . Theor Appl Genet . 2004 ; 108 : 1492 ± 1502 . https://doi.org/10.1007/ s00122-004 -1590-3 PMID: 14968307
24. Yu SL , Pan LJ , Yang QL , Min P , Ren ZK , Zhang HS . Comparison of the Δ12 fatty acid desaturase gene between high-oleic and normal-oleic peanut genotypes . J Genet Genomics . 2008 ; 35 : 1 ± 7 .
25. Wang CT , Tang YY , Wang XZ , Zhang SW , Li GJ , Zhang JC , et al. Sodium azide mutagenesis resulted in a peanut plant with elevated oleate content . Electron J Biotechnol . 2011a ; 14 ( 2 ): http://dx.doi.org/10. 2225/vol14-issue2 - fulltext-4.
26. Fang CQ , Wang CT , Wang PW , Tang YY , Wang XZ , Cui FG , et al. Identification of a novel mutation in FAD2B from a peanut EMS mutant with elevated oleate content . J Oleo Sci . 2012 ; 61 ( 3 ): 143 ± 8 . PMID: 22362145
27. Chu Y , Wu CL , Holbrook CC , Tillman BL , Person G , Ozias±Akins P. Marker-assisted selection to pyramid nematode resistance and the high oleic trait in peanut . Plant Genome . 2011 ; 4 ( 2 ): 110 ± 7 .
28. Barkley NA , Chenault±Chamberli KD , Wang ML , Pittman RN . Development of a real time PCR genotyping assay to identify high oleic acid peanuts (Arachis hypogaea L.) . Mol Breeding . 2010 ; 25 : 541 ± 8 .
29. Barkley NA , Wang ML , Pittman RN. A real-time PCR genotyping assay to detect FAD2A SNPs in peanuts (Arachis hypogaea L.) . Electron J Biotechnol . 2011 ; 14 ( 1 ): http://dx.doi.org/10.2225/vol13-issue1 - fulltext-12.
30. Chen ZB , Wang ML , Barkley NA , Pittman RN . A simple allele-specific PCR assay for detecting FAD2 alleles in both A and B genomes of the cultivated peanut for high-oleate trait selection . Plant Mol Biol Rep . 2010 ; 28 : 542 ± 8 .
31. Stalker HT , Dhesi JS , Parry DC , Hahn JH . Cytological and interfertility relationships of Arachis section Arachis . Am J Bot . 1991 ; 78 : 238 ± 46 .
32. Kochert G , Stalker HT , Gimenes M , Galgaro L , Lopes CR , Moore K. RFLP and cytogenetic evidence on the origin and evolution of allotetraploid domesticated peanut, Arachis hypogaea (Leguminosae) . Am J Bot . 1996 , 83 : 1282 ± 91 .
33. Chen X , Li H , Pandey MK , Yang Q , Wang X , Garg V , et al. Draft genome of the peanut A-genome progenitor (Arachis duranensis) provides insights into geocarpy, oil biosynthesis, and allergens . Proc Natl Acad Sci U S A . 2016 ; 113 ( 24 ): 6785 ± 90 . https://doi.org/10.1073/pnas.1600899113 PMID: 27247390
34. Bertioli DJ , Cannon SB , Froenicke L , Huang GD , Farmer AD , Cannon EKS , et al. The genome sequences of Arachis duranensis and Arachis ipaensis, the diploid ancestors of cultivated peanut . Nature GeNetics . 2016 ; 48 ( 4 ): 438 ± 46 . https://doi.org/10.1038/ng.3517 PMID: 26901068
35. Hall TA . BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95 /98/NT. Nucl Acids Symp Ser. 1999 ; 41 : 95 ± 8 .
36. Sayanova O , Beaudoin F , Libisch B , Castel A , Shewry PR , Napier JA . Mutagenesis and heterologous expression in yeast if a plant Δ6 fatty acid desaturase . J Exp Bot . 2001 ; 52 : 1581 ± 5 . PMID: 11457919
37. Gao JP , Ajjawi I , Manoli A , Sawin A , Xu CC , Froehlich JE , et al. FATTY ACID DESATURASE4 of Arabidopsis encodes a protein distinct from characterized fatty acid desaturases . The Plant Journal . 2009 ; 60 : 832 ±9. https://doi.org/10.1111/j. 1365 - 313X . 2009 . 04001 . x PMID : 19682287
38. Shanklin J , Whittle E , Fox BG . Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase . Biochemistry . 1994 ; 33 : 12787 ± 94 . PMID: 7947684
39. Shanklin J , Cahoon EB . Desaturation and related modifications of fatty acids . Annu Rev Plant Physiol Plant Mol Biol . 1998 ; 49 : 611 ± 41 . https://doi.org/10.1146/annurev. arplant.49.1.611 PMID: 15012248
40. Sato N , Moriyama T. Genomic and biochemical analysis of lipid biosynthesis in the unicellular Rhodophyte Cyanidioschyzon merolae: Lack of a plastidic desaturation pathway results in the coupled pathway of galactolipid synthesis . Eukaryotic Cell . 2007 ; 6 : 1006 ± 17 . https://doi.org/10.1128/EC.00393-06 PMID: 17416897
41. Yang L , Chen J , Huang C , Liu Y , Jia S , Pan L , et al. Validation of a cotton-specific gene, Sad1, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic cottons . Plant Cell Rep . 2005 ; 24 : 237 ± 45 . https://doi.org/10.1007/s00299-005 -0929-9 PMID: 15726375
42. Thomson TM , Lozano JJ , Loukili N , Carrio R , Serras F , Cormand B , et al. Fusion of the human gene for the polyubiquitination coeffector UEV1 with Kua, a newly identified gene . Genome Res . 2000 ; 10 : 1743 ± 56 . PMID: 11076860
43. Alonso DL , Maroto FG , Ruiz JR , Garrido JA , Vilches MA . Evolution of the membrane-bound fatty acid desaturases . Biochemical Systematics and Ecology . 2003 ; 31 : 1111 ± 24 .
44. Chi XY , Hu RB , Yang QL , Zhang XW , Pan LJ , Chen N , et al. Validation of reference genes for gene expression studies in peanut by quantitative real-time RT-PCR . Mol Genet Genomics . 2012 ; 287 : 167 ± 76 . https://doi.org/10.1007/s00438-011 -0665-5 PMID: 22203160
45. Michaelson LV , ZaÈuner S , Markham JE , Haslam RP , Desikan R , Mugford S , et al. Functional characterization of a higher plant sphingolipid Δ4-desaturase: defining the role of sphingosine and sphingosine-1- phosphate in Arabidopsis . Plant Physiology . 2009 ; 149 : 487 ± 98 . https://doi.org/10.1104/pp. 108 .129411 PMID: 18978071
46. Zhang J , Liu H , Sun J , Li B , Zhu Q , Chen S , et al. Arabidopsis fatty acid desaturase FAD2 is required for salt tolerance during seed germination and early seedling growth . PLoS ONE . 2012 ; 7:e30355 . https:// doi.org/10.1371/journal.pone. 0030355 PMID: 22279586
47. Zhang JT , Zhu JQ , Zhu Q , Liu H , Gao XS , Zhang HX . Fatty acid desaturase-6 (Fad6) is required for salt tolerance in Arabidopsis thaliana . Biochem Biophys Res Commun . 2009 ; 390 : 469 ± 74 . https://doi.org/ 10.1016/j.bbrc. 2009 . 09 .095 PMID: 19799856
48. Chen M , Markham JE , Cahoon EB . Sphingolipid Δ8 unsaturation is important for glucosylceramide biosynthesis and low-temperature performance in Arabidopsis . Plant J. 2012 ; 69 : 769 ± 81 . https://doi.org/ 10.1111/j. 1365 - 313X . 2011 . 04829 . x PMID : 22023480
49. Kachroo A , Shanklin J , Whittle E , Lapchyk L , Hildebrand D , Kachroo P. The Arabidopsis stearoyl -acyl carrier protein-desaturase family and the contribution of leaf isoforms to oleic acid synthesis . Plant Mol Biol . 2007 ; 63 : 257 ± 71 . https://doi.org/10.1007/s11103-006-9086-y PMID: 17072561
50. Fukuchi±Mizutani M , Tasaka Y , Tanaka Y , Ashikari T , Kusumi T , Murata N. Characterization of Δ9 acyllipid desaturase homologues from Arabidopsis thaliana . Plant Cell Physiol . 1998 ; 39 ( 2 ): 247 ± 53 . PMID: 9559566
51. Smith MA , Dauk M , Ramadan H , Yang H , Seamons LE , Haslam RP , et al. Involvement of Arabidopsis ACYL-COENZYME A DESATURASE-LIKE2 (At2g31360) in the biosynthesis of the very long chain monounsaturated fatty acid components of membrane lipids . Plant Physiology . 2013 ; 161 : 81 ± 96 . https://doi.org/10.1104/pp. 112 .202325 PMID: 23175755
52. Nishiuchi T , Nakamura T , Abe T , Kodama H , Nishimura M , Iba K. Tissue-specific and light-responsive regulation of the promoter region of the Arabidopsis thaliana chloroplast ω-3 fatty acid desaturase gene (FAD7) . Plant Mol Biol . 1995 ; 29 : 599 ± 609 . PMID: 8534855
53. Liu W , Li W , He QL , Daud MK , Chen JH , Zhu SJ . Characterization of 19 genes encoding membranebound fatty acid desaturases and their expression profiles in Gossypium raimondii under low temperature . PLoS ONE . 2015 ; 10 ( 4 ):e0123281. https://doi.org/10.1371/journal.pone. 0123281 PMID: 25894196
54. Dong CJ , Cao N , Zhang ZG , Shang QM . Characterization of the fatty acid desaturase genes in cucumber: structure, phylogeny, and expression patterns . PLoS ONE . 2016 ; 11 ( 3 ):e0149917. https://doi.org/ 10.1371/journal.pone. 0149917 PMID: 26938877
55. Lee KR , Lee Y , Kim EH , Lee SB , Roh KH , Kim JB , et al. Functional identification of oleate 12-desaturase and ω-3 fatty acid desaturase genes from Perilla frutescens var . frutescens. Plant Cell Rep . 2016 ; 35 : 2523 ± 37 . https://doi.org/10.1007/s00299-016 -2053-4 PMID: 27637203
56. Singh SC , Sinha RP , HaÈder DP . Role of lipids and fatty acids in stress tolerance in cyanobacteria . Acta Protozool . 2002 ; 41 : 297 ± 308 .
57. Swan TM , Watson K. Membrane fatty acid composition and membrane fluidity as parameters of stress tolerance in yeast . Can J Microbiol . 1997 ; 43 : 70 ± 7 . PMID: 9057297
58. Mikami K , Murata N. Membrane fluidity and the perception of environmental signals in cyanobacteria and plants . Progress in Lipid Research . 2003 ; 42 : 527 ± 43 . PMID: 14559070
59. Chapman D. Phase transitions and fluidly characteristics of lipids and cell membranes . Quart Rev Biophys . 1975 ; 8 : 185 ± 235 .
60. Los DA , Murata N. Structure and expression of fatty acid desaturases . Biochim Biophys Acta . 1998 ; 1394 :3± 15 . PMID: 9767077
61. Chen M , Thelen JJ . ACYL-LIPID DESATURASE2 is required for chilling and freezing tolerance in Arabidopsis . Plant Cell . 2013 ; 25 ; 1430 ± 44 . https://doi.org/10.1105/tpc.113.111179 PMID: 23585650
62. Khodakovskaya M , McAvoy R , Peters J , Wu H , Li Y. Enhanced cold tolerance in transgenic tobacco expressing a chloroplast ω-3 fatty acid desaturase gene under the control of a cold-inducible promoter . Planta . 2006 ; 223 : 1090 ± 100 . https://doi.org/10.1007/s00425-005 -0161-4 PMID: 16292565
63. Im YJ , Han O , Chung GC , Cho BH . Antisense expression of an Arabidopsis omega-3 fatty acid desaturase gene reduces salt/drought tolerance in transgenic tobacco plants . Mol Cells . 2002 ; 13 : 264 ± 71 . PMID: 12018849
64. Zhang M , Barg R , Yin M , Gueta-Dahan Y , Leikin-Frenkel A , Salts Y , et al. Modulated fatty acid desaturation via overexpression of two distinct ω-3 desaturases differentially alters tolerance to various abiotic stresses in transgenic tobacco cells and plants . Plant J . 2005 ; 44 : 361 ± 71 . https://doi.org/10.1111/j. 1365 - 313X . 2005 . 02536 . x PMID : 16236147
65. Liu HL , Shen HT , Chen C , Zhou XR , Liu H , Zhu JB . Identification of a putative stearoyl acyl-carrier-protein desaturase gene from Saussurea involucrate . Biologia plantarum . 2015 ; 59 ( 2 ): 316 ± 24 .
66. Guan LL , Wu W , Hu B , Li D , Chen JW , Hou K , et al. Devolopmental and growth temperature regulation of omega-3 fatty acid desaturase genes in safflower (Carthamus tinctorius L.) . Genetics and Molecular Research . 2014 ; 13 ( 3 ): 6623 ± 37 . https://doi.org/10.4238/ 2014 .August. 28 .7 PMID: 25177943
67. Nair PMG , Kang I , Moon B , Lee C. Effects of low temperature stress on rice (Oryza sativa L.) plastid ω- 3 desaturase gene, OsFAD8 and its functional analysis using T-DNA mutants . Plant Cell Tiss Organ Cult . 2009 ; 98 : 87 ± 96 .
68. Wang HS , Yu C , Tang XF , Zhu ZJ , Ma NN , Meng QW . A tomato endoplasmic reticulum (ER)-type omega-3 fatty acid desaturase (LeFAD3) functions in early seedling tolerance to salinity stress . Plant Cell Rep . 2014 ; 33 : 131 ± 42 . https://doi.org/10.1007/s00299-013-1517-z PMID: 24129846
69. Liu XY , Yang JH , Li B , Yang XM , Meng QW . Antisense-mediated depletion of tomato chloroplast omega-3 fatty acid desaturase enhances thermal tolerance . J Integr Plant Biol . 2006 ; 48 : 1096 ± 107
70. RomaÂn AÂ , Andreu V , HernaÂndez ML , Lagunas B , Picorel R , MartÂõnez-Rivas JM , et al. Contribution of the different omega-3 fatty acid desaturase genes to the cold response in soybean . J Exp Bot . 2012 ; 63 : 4973 ± 82 . https://doi.org/10.1093/jxb/ers174 PMID: 22865909
71. Domergue F , Spiekermann P , Lerchl J , Beckmann C , Kilian O , Kroth PG , et al. New insight into Phaeodactylum tricornutum fatty acid metabolism. Cloning and functional characterization of plastidial and microsomal Δ12-fatty acid desaturases . Plant Physiol . 2003 ; 131 : 1648 ± 60 . https://doi.org/10.1104/pp. 102 .018317 PMID: 12692324
72. Ray TK , Holly SP , Knauft DA , Abbott AG , Powell GL . The primary defect in developing seed from the high oleate variety of peanut (Arachis hypogaea L.) is the absence of 12-desaturase activity . Plant Sci . 1993 ; 91 : 15 ± 22 .
73. Li SF , Song LY , Yin WB , Chen YH , Chen L , Li JL , et al. Isolation and functional characterization of the genes encoding Δ8±sphingolipid desaturase from Brassica rapa . Journal of Genetics and Genomics . 2012 ; 39 : 47e59 .
74. ZaÈuner S , Ternes P , Warnecke D. Biosynthesis of sphingolipids in plants (and some of their functions) . Adv Exp Med Biol . 2010 ; 688 : 249e263 .
75. Moreno-PeÂrez AJ , MartÂõnez±Force E , GarceÂs R , Salas JJ . Sphingolipid base modifying enzymes in sunflower (Helianthus annuus): cloning and characterization of a C4-hydroxylase gene and a new paralogous Δ8-desaturase gene . J. Plant Physiol . 2011 ; 168 : 831e839 .
76. Thompson JD , Higgins DG , Gibson TJ . CLUSTALW: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice . Nucleic Acids Res . 1994 ; 22 : 4673 ± 80 . PMID: 7984417
77. Tamura K , Dudley J , Nei M , Kumar S. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0 . Molecular Biology and Evolution. 2007 ; 24 : 1596 ±9. https://doi.org/10.1093/molbev/ msm092 PMID: 17488738
78. Livak KJ , Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2(- Delta Delta C(T)) Method Methods . 2001 ; 25 : 402 ±8. https://doi.org/10.1006/meth. 2001 . 1262 PMID: 11846609
79. Browse J , McCourt PJ , Somerville CR . Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue . Anal Biochem . 1986 ; 152 : 141 ± 5 . PMID: 3954036
80. Markham JE , Li J , Cahoon EB , Jaworski JG . Separation and identification of major plant sphingolipid classes from leaves . Journal of Biological Chemistry . 2006 ; 281 : 22684 ± 94 . https://doi.org/10.1074/jbc. M604050200 PMID: 16772288
81. Merrill AH , Caligan TB , Wang E , Peters K , Ou J . Analysis of sphingoid bases and sphingoid base 1- phosphates by high performance liquid chromatography . Methods in Enzymology . 2000 , 312 :3± 9 . PMID: 11070857