Characterization of 19 Genes Encoding Membrane-Bound Fatty Acid Desaturases and their Expression Profiles in Gossypium raimondii Under Low Temperature
Characterization of 19 Genes Encoding Membrane-Bound Fatty Acid Desaturases and their Expression Profiles in Gossypium raimondii Under Low Temperature
Wei Liu 0 1
Wei Li 0 1
Qiuling He 0 1
Muhammad Khan Daud 0 1
Jinhong Chen 0 1
Shuijin Zhu 0 1
0 1 Department of Agronomy, Zhejiang University , Hangzhou, 310058, China , 2 Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology , Kohat, 26000, Pakistan , 3 Jiangsu Collaborative Innovation Center for Modern Crop Production , Nanjing, 210095 , China
1 Academic Editor: Xianlong Zhang, National Key Laboratory of Crop Genetic Improvement , CHINA
To produce unsaturated fatty acids, membrane-bound fatty acid desaturases (FADs) can be exploited to introduce double bonds into the acyl chains of fatty acids. In this study, 19 membrane-bound FAD genes were identified in Gossypium raimondii through database searches and were classified into four different subfamilies based on phylogenetic analysis. All 19 membrane-bound FAD proteins shared three highly conserved histidine boxes, except for GrFAD2.1, which lost the third histidine box in the C-terminal region. In the G. raimondii genome, tandem duplication might have led to the increasing size of the FAD2 cluster in the Omega Desaturase subfamily, whereas segmental duplication appeared to be the dominant mechanism for the expansion of the Sphingolipid and Front-end Desaturase subfamilies. Gene expression analysis showed that seven membrane-bound FAD genes were significantly up-regulated and that five genes were greatly suppressed in G. raimondii leaves exposed to low temperature conditions.
Competing Interests: The authors have declared
that no competing interests exist.
Both saturated and unsaturated fatty acids are major components of membrane phospholipids
in plants as well as triacylglycerols in seeds. Unsaturated fatty acids usually contain one or more
double bonds in their acyl chains. The number and position of double bonds in fatty acids
profoundly influence their physical and physiological properties [1,2]. The desaturation of fatty
acids is catalyzed by a class of enzymes called fatty acid desaturases (FADs) [1,3]. The two major
groups of fatty acid desaturases, soluble and membrane-bound, have been identified and have no
evolutionary relationship with each other [4,5]. The soluble desaturases have two conserved
histidine boxes and are represented by the plant stearoyl-ACP desaturase, which specifically
desaturates stearoyl-ACP (18:0) to produce ACP-bound oleic acid (18:1) . The membrane-bound
desaturases contain three histidine boxes and are ubiquitous in prokaryotes and eukaryotes [4,5].
They comprise a highly diversified family that includes many different types of regioselectivities,
such as 4, 5, 6, 7, 8, 9, 12 and 15 [5,7].
Most of the fatty acids residing in plant membranes are unsaturated. Their level of
unsaturation is highly dependent upon the tolerance of a given plant for various environmental stresses,
especially temperature stress [8,9,10]. Previous studies have revealed that genes encoding
membrane-bound FAD proteins are crucial for the sustenance of plants faced with different
environmental stresses. In rice, OsFAD8 has been reported to have a functional role in stress tolerance
at low temperatures . FAD2 and FAD6 were found to be active in seedlings of Arabidopsis
under salinity stress [12,13]. The ads2 mutant Arabidopsis plants showed increased sensitivity to
chilling and freezing temperatures , and the mutants of SLD genes also showed enhanced
sensitivity to prolonged low-temperature exposure . In tomato, LeFAD3 over-expression
enhanced the tolerance of tomato seedlings for salinity stress , whereas silencing the LeFAD7
gene alleviated high-temperature stress . In transgenic tobacco plants, over-expressing
FAD7 also showed enhanced cold tolerance , whereas antisense expression of the
Arabidopsis FAD7 reduced salt and drought tolerance . In soybean, the expression of FAD3 and
FAD7 was tightly regulated in response to cold temperature .
Cotton is the major source of natural fibers used in the textile industry. It is also a promising
oilseed crop. Cotton is mostly grown in tropical and subtropical regions of the world, and its
cultivation has been achieved even in relatively cold regions. Low temperature (under 15C) can
adversely affect plant development, resulting in poor germination and higher seedling mortality
due to disease infection, which ultimately cause significant losses in yield . Although the
plant has been grown in cold areas, little is known about the molecular responses of cotton to
low temperature. The 12 desaturases (FAD2) were extensively characterized in Gossypium
hirsutum [21,22,23,24], and expression analysis suggested that FAD2 genes play a direct role in
cotton adaptation to cold stress . More recently, 15 fatty acid desaturases (FAD3 and FAD7/8)
were identified in Gossypium, and one of the genes, termed FAD7/8-1, was dramatically induced
during cold temperature treatment of G. hirsutum seedlings .
Gossypium raimondii is a diploid cotton species, whose progenitor is the putative contributor
of the D subgenome to the economically important fiber-producing cotton species G. hirsutum
and G. barbadense . Sequencing of the G. raimondii genome has provided an opportunity
for genome-wide analysis of all the genes belonging to specific gene families in cotton. In this
paper, our main objectives were to identify membrane-bound FAD genes in G. raimondii
through homology searches, to classify them into different subfamilies according to
phylogenetic analysis, as well as to investigate their expression profiles in different tissues and under a cold
stress regime. The results may provide information valuable for understanding the biological
roles of membrane-bound FAD genes in the response of cotton to cold stress, and may also help
cotton breeders improve the quality of cotton oil via molecular design breeding.
Materials and Methods
Database search and gene retrieval
The genome database (release v2.1)  of G. raimondii was downloaded from Phytozome
(http://www.phytozome.net/). Seventeen membrane-bound fatty acid desaturases of
Arabidopsis (S1 Table) [28,29] were obtained from the Arabidopsis Information Resource (TAIR release
10, http://www.arabidopsis.org/). To identify all candidate membrane-bound FAD genes of
G. raimondii, these FAD protein sequences of Arabidopsis were employed as queries to search
the G. raimondii genome database using BlastP and tBlastN programs with default
parameters. Subsequently, the Pfam (http://pfam.sanger.ac.uk/search)  and SMART databases
(http://smart.embl-heidelberg.de/)  were used to confirm each putative member of the
FAD family. The theoretical Mw (molecular weight) and pI (isoelectric point) of the full-length
protein were predicted using the ProtParam tool (http://web.expasy.org/protparam/).
Multiple sequence alignment and phylogenetic analysis
Multiple sequence alignments of full-length protein sequences were performed using Clustal X
version 2.0  with default parameters. The Neighbor-Joining phylogenetic trees were
constructed using MEGA 5.2  with pairwise deletion option and poisson correction model.
Bootstrap tests were carried out with 1000 replicates for statistical reliability.
Gene structures, chromosomal locations and gene duplications
To illustrate exon-intron organization for an individual gene, the Gene Structure Display
Server (GSDS, http://gsds1.cbi.pku.edu.cn/)  was employed to compare the predicted coding
sequences (CDSs) with their corresponding genomic sequences.
The location data of all membrane-bound FAD genes were acquired from the genome
annotation document. The chromosome location image of membrane-bound FAD genes was
generated using MapInspect software according to their starting positions on the G. raimondii
Gene duplication of membrane-bound FAD genes in G. raimondii was defined according to
(1) the length of aligned sequence cover was > 80% of the longer gene, (2) the identity of the
aligned regions was > 80%, and (3) only one duplication event was counted for tightly linked
genes [36,37,38]. With the chromosomal locations of membrane-bound FAD genes, two types
of gene duplications were recognized (i.e., tandem and segmental duplications).
Plant materials and low temperature stress treatment
All the plants of G. raimondii were grown in a temperature-controlled chamber at 28C
with a photoperiod of 16 hours light and 8 hours dark. After ten days, the leaves, stems,
roots, and cotyledons of some seedlings were sampled to analyze tissue-specific expression.
To examine the expression patterns of membrane-bound FAD genes under low
temperature stress, the plant leaves of the remaining seedlings treated at 10C in the
temperaturecontrolled chamber were harvested at 0, 3, 6, and 12 hours, which represented normal
plants, slight stress, moderate stress, and severe stress, respectively. All collected samples
were immediately frozen in liquid nitrogen and stored at -80C. Three biological replicates
were conducted per sample.
RNA isolation and quantitative real-time RT-PCR (qRT-PCR)
Total RNA was extracted from all samples using the EASYspin Plus Total RNA Extraction
Kit (Aidlab, Beijing, China), and first-strand cDNAs were synthesized with the PrimeScript
1st Strand cDNA Synthesis Kit (TakaRa, Dalian, China) according to the manufacturers
protocols. For quantitative real-time RT-PCR (qRT-PCR) assay, gene-specific primers
were designed for the membrane-bound FAD genes according to their CDSs (S2 Table).
The qRT-PCR was performed with the SYBR Premix Ex Taq (TakaRa, Dalian, China) in
the BioRad CFX96 Real-time PCR System following the manufacturers instructions. The
cotton UBQ7 gene was used as an internal reference for all the qRT-PCR analyses. Each
sample was performed in three biological replicates. The relative expression levels were
calculated according to the 2-CT method . The expression profiles were clustered using
the Cluster 3.0 software .
The candidate membrane-bound FAD genes were identified from the G. raimondii genome
using the BlastP and tBlastN programs with the query sequences of Arabidopsis
membranebound FAD genes. The retrieved sequences were submitted to the Pfam and SMART databases
to confirm the presence of conserved domains (Pfam: PF00487). As a result, 19 non-redundant
membrane-bound FAD genes were confirmed in G. raimondii. For comparative analysis, the
membrane-bound FAD genes in rice (S1 Table) were also identified from the Rice Genome
Annotation Project Database (RGAP release 7, http://rice.plantbiology.msu.edu/index.shtml)
following the same strategy. All identified FAD genes were named according to their orthology
with reported counterparts in Arabidopsis. Detailed information about the 19
membranebound FAD genes in G. raimondii is provided in Table 1. The protein sequences encoded by
these 19 FAD genes varied in length from 292 amino acids for GrFAD2.1 to 477 amino acids
for GrFAD8.2, with an average of approximately 397 amino acids. The predicted molecular
weight (Mw) of these proteins ranged from 33.44 kDa to 55.30 kDa, and the theoretical
isoelectric point (pI) ranged from 6.95 to 9.61.
Phylogenetic analysis of membrane-bound FAD genes
To evaluate the phylogenetic relationships of membrane-bound FAD genes in different species,
an unrooted phylogenetic tree was constructed according to the alignments of full-length
protein sequences of membrane-bound FADs in G. raimondii, Arabidopsis, and rice. In previous
reports, membrane-bound desaturases from eukaryotic genomes were divided into four
functional subfamilies: First Desaturase, Omega Desaturase, Front-end Desaturase, and
Sphingolipid Desaturase . As shown in the phylogenetic tree (Fig 1), all of the membrane-bound
desaturases used in this study fell into these four subfamilies.
Fig 1. Phylogenetic relationships and motif compositions of membrane-bound FAD genes from G. raimondii, Arabidopsis and rice. Unrooted
phylogenetic tree (left panel): Four subfamilies marked with different color backgrounds are labeled as Omega, Sphingolipid, Front-end and First. Motif
compositions (right panel): Protein sequences are indicated by thick gray lines, and the conserved motifs are represented by different colored boxes. The
length (amino acids) of the protein and motif can be estimated using the scale bar at the top.
The First Desaturase subfamily included 7 desaturases and 9 desaturases, encoded by
ADS genes, which generally introduced the first double bond into the saturated acyl chain
[7,41]. There were nine members of the subfamily in Arabidopsis, but only one gene was found
in G.raimondii, which was a homolog of AtADS3 (also termed AtFAD5).
The Omega Desaturase subfamily contained 12 desaturases and 15 desaturases,
which introduce a double bond between an existing double bond and the acyl end .
The 12 desaturases encoded by FAD2 and FAD6 were frequently called 6 desaturases
[42,43], and the 15 desaturases encoded by FAD3, FAD7 and FAD8 were also called 3
desaturases [44,45,46]. In the phylogenetic tree, FAD2 and FAD6 genes were grouped in
separate branches. G. raimondii, Arabidopsis and rice had one FAD6 gene each, but the
number of FAD2 genes was diverse, with an expanded number (up to five) in G. raimondii,
which was greater than that in Arabidopsis (one) and rice (three). FAD3, FAD7, and FAD8
formed the other cluster, which contained three FAD genes from Arabidopsis, four from
rice, and five from G. raimondii.
The Front-end Desaturase subfamily was comprised of sphingolipid 8 desaturases, which
were encoded by SLD genes [7,15]. Five SLD genes were found in G. raimondii, two SLD genes
in Arabidopsis, and one SLD gene in rice.
The last group was the Sphingolipid Desaturase subfamily, which was represented by
sphingolipid 4 desaturases [7,47]. The group contained one gene from Arabidopsis, one gene from
rice, and two genes from G. raimondii.
Interestingly, the number of members identified in G. raimondii was greater than that
in Arabidopsis and rice in three of four subfamilies (Table 2). This result suggested that the
membrane-bound FAD genes in the G. raimondii genome might have undergone
species-specific expansion over the course of evolution.
Conserved motifs in membrane-bound FAD genes
The membrane-bound desaturases shared three highly conserved histidine boxes, which were
thought to be involved in the formation of the active site of each desaturase [1,48]. All of the
membrane-bound FAD proteins analyzed in this study contained these three histidine boxes,
except for GrFAD2.1, which lost the third histidine box in the C-terminal region (Fig 1, S3
Table). Additionally, the relative positions of the three histidine boxes in the protein sequences
were similar among desaturases. The first and second histidine boxes were located near each
other, with only 31 or 32 amino acid residues between them. The intervening length in the
First and Omega Desaturase subfamilies was 31 amino acid residues, and there were 32 amino
acid residues in the Front-end and Sphingolipid Desaturase subfamilies. The third histidine
box was positioned in the C-terminal region of the proteins. The number of amino acid
residues between the second and third histidine boxes was different among subfamilies, but in
each subfamily or cluster, the number was almost identical. For example, there were 127 amino
Fig 2. Chromosomal localization of membrane-bound FAD genes in G. raimondii. Chromosome numbers are indicated above each vertical bar. The
scale is in megabases (Mb). The black lines connecting the genes indicate segmental duplications, and tandem duplications are marked by red lines.
acid residues in the First Desaturase subfamily and 161 or 162 amino acid residues in the
FAD3/FAD7/FAD8 cluster of the Omega Desaturase subfamily.
To further confirm the conservation of amino acid residues in the histidine boxes, the
sequence logos of the three histidine boxes in each subfamily were generated using the WebLogo
program (S1 Fig). As previously reported [49,50,51], it was observed that the first residue in the
third histidine box was glutamine rather than histidine in the Front-end Desaturase subfamily.
Apart from this divergence, the remaining histidines were strongly conserved among
subfamilies. However, the other amino acids in the histidine boxes differed greatly. Remarkably, there
were four amino acid residues between the histidines in the first histidine-box of the First
Desaturase subfamily, but only three in the other three subfamilies.
FAD2, known as the ER-localized membrane-bound FAD, contained an ER (endoplasmic
reticulum) retrieval motif consisting of F-X-X-K/R/D/E-F (F are large hydrophobic amino acid
residues such as F/Y/W/I/L/V) at the C-terminus . Expectedly, all five FAD2 proteins identified in
G. raimondii had the ER retrieval motif (Fig 1). The motif was YHNKF in GrFAD2.1, YRNKF in
GrFAD2.2, FRNKL in GrFAD2.3, FRNKL in GrFAD2.4, and FRNKI in GrFAD2.5, respectively.
SLD, which functions as a sphingolipid 8 desaturase, was characterized by the presence of an
Nterminal cytochrome b5 domain . Through searches in Pfam database, it was found that all five
SLD genes in G. raimondii contained the cytochrome b5 domain at the N-terminus (Fig 1).
Chromosomal locations and structure of membrane-bound FAD genes
The 19 membrane-bound FAD genes were mapped to the 11 chromosomes in G. raimondii
(Fig 2). They were distributed unevenly among the chromosomes. Chromosomes 2 and 13
contained three membrane-bound FAD genes each and chromosomes 1, 7, 9, and 11 contained
two genes each, while only a single FAD gene was localized on each of the chromosomes 4, 6, 8,
10, and 12. There were no FAD genes located on chromosomes 3 and 5. Four duplicated gene
pairs, i.e., GrFAD2.3/GrFAD2.4, GrDSD1/GrDSD2, GrSLD1/GrSLD2, and GrSLD4/GrSLD5,
were found in the G. raimondii genome. According to the chromosomal distribution of the
membrane-bound FAD genes, three duplication events were assigned to the segmental
duplication. GrFAD2.3 and GrFAD2.4, which were positioned adjacently on chromosome 2 with no
intervening genes, were involved in a tandem duplication event.
A separate phylogenetic tree was generated using the protein sequences of all
membranebound FAD genes identified in G. raimondii and the exon-intron structures of these genes
were compared (Fig 3). All members of the FAD3/FAD7/FAD8 cluster contained eight exons.
Their conserved gene structure supported their close evolutionary relationship. Most of the
FAD2 genes, including the duplicated genes GrFAD2.3 and GrFAD2.4, had only one exon, with
the exception of GrFAD2.1, which contained three exons. For SLD, the genes of two duplicated
pairs, GrSLD1/GrSLD2 and GrSLD4/GrSLD5, had the same gene structures and contained one
exon. GrDSD1 and GrDSD2, another duplicated gene pair, contained two exons. However,
GrSLD3 had three exons, GrFAD5 had five exons, and GrFAD6 contained up to ten exons.
Expression of the membrane-bound FAD genes in different tissues
To investigate the expression profiles of membrane-bound FAD genes in different tissues of G.
raimondii, qRT-PCR analysis was performed to examine the gene expression levels in the
roots, stems, cotyledons, and leaves of 10-day-old seedlings. Because no transcripts could be
detected for GrFAD2.1 in the four representative tissues using up to five gene-specific primers
in reverse transcription (RT)-PCR analysis (data not shown), this gene was not included in the
qRT-PCR analysis. As shown in Fig 4, the expression patterns of the 18 membrane-bound
FAD genes varied significantly in the tissues analyzed in this study. 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. These results demonstrated that the majority of these
membrane-bound FAD genes exhibited tissue-specific expression patterns, which was consistent
with membrane-bound FAD genes in Arabidopsis and soybean, which also showed specific
spatial expression patterns .
Furthermore, the tissue-specific expression patterns of the genes involved in duplication
events were compared. Although the genes in all four duplicated gene pairs shared high
Fig 3. Phylogenetic relationship and gene structure of membrane-bound FAD genes in G. raimondii. Exons are represented by white boxes and
introns by black lines.
Fig 4. Expression patterns of membrane-bound FAD genes in four representative tissues of G.
raimondii seedlings. The color scale represents the gene expression intensity, green indicating low levels of
transcript abundance and red indicating high transcript abundance.
sequence similarity and the same gene structure, the gene expression patterns were highly
diverse. For instance, GrFAD2.3 showed preferential expression in the stem and cotyledon, but
that in the root and leaf was limited. And GrFAD2.4, which was involved in a duplication event
with GrFAD2.3, showed preferential expression in the stem and leaf.
The expression patterns of the G. raimondii membrane-bound FAD genes in the leaves of
10-day-old seedlings under low temperature stress (10C) were investigated in this study, and
all the gene expression levels responsive to slight (3 hours), moderate (6 hours), and severe (12
hours) cold stresses were compared with those of normal plants as shown in Fig 5, with the
exception of GrFAD2.1. Out of the 18 membrane-bound FAD genes, seven genes, i.e., GrFAD8.1,
GrFAD2.2, GrFAD8.2, GrSLD2, GrSLD4, GrDSD1, and GrSLD5, showed a marked increase in
transcript level when treated with cold stress. Among them, the expression levels of GrFAD8.1
and GrSLD5 were higher at 12 hours after cold treatment, and GrSLD2 and GrSLD4 almost
reached their highest levels at 6 hours after cold treatment, whereas GrFAD2.2, GrFAD8.2, and
GrDSD1 were expressed highly at 3 hours of cold treatment. Additionally, the expression levels
of 5 other genes, i.e., GrFAD2.4, GrFAD3.1, GrFAD3.2, GrFAD2.5, and GrDSD2, were slightly
up-regulated in response to cold stress. In contrast, the five genes GrFAD5, GrFAD7, GrFAD2.3,
Fig 5. Expression profiling of membrane-bound FAD genes in leaves under 10C treatment. The color
scale represents the gene expression intensity, green indicating low levels of transcript abundance and red
indicating high transcript abundance.
GrSLD1, and GrSLD3 were significantly down-regulated after long periods of cold stress
treatment, and one gene, GrFAD6, was suppressed slightly during treatment with cold stress.
There were also some differences in expression patterns between duplicated genes under low
FAD genes encode the enzymes that catalyze the desaturation of fatty acids, which affect the
oxidative stability and nutritional value of seed storage oils . Many studies have indicated that
modifying the activity of FAD genes could create transgenic soybean lines with improved seed
oil quality through altering relative amounts of fatty acids [54,55,56,57]. Cotton is also a
significant oilseed crop, and cottonseeds are an important source of livestock feed, foodstuff and oil
. FAD2 genes have already been genetically manipulated for cottonseed oil improvement
[59,60,61]. However, only several FAD genes encoding the 12 and 15 desaturases have been
characterized in cotton [22,23,24,25]. In this study, a comprehensive set of 19 non-redundant
membrane-bound fatty acid desaturases was identified from the available genome sequences of
G. raimondii. Undoubtedly, the 19 membrane-bound FAD genes identified in the diploid cotton
will provide candidate genes for the gene engineering of fatty acid biosynthesis in cotton.
The FAD genes were named based on their orthologous genes in Arabidopsis. However,
following this nomenclature, it was difficult to distinguish FAD7 and FAD8, due to their high degree
of homology. It has been demonstrated that FAD7 was highly expressed at high temperatures
[11,62] and that the transcript level of FAD8 increased at low temperatures [11,62,63,64].
Therefore, according to the expression patterns of FAD genes under low temperature stress analyzed
in this study, GrFAD7 and two isoforms of GrFAD8 (GrFAD8.1 and GrFAD8.2) were
designated. GrFAD7 was suppressed at low temperature, whereas GrFAD8.1 was consistently induced
under long duration cold exposure and GrFAD8.2 was rapidly induced at 3 hours under low
Analysis of protein structure showed that all of the 19 membrane-bound desaturases in G.
raimondii, except for GrFAD2.1, had the three highly conserved histidine boxes, which
contained strongly conserved histidine residues. GrFAD2.1 contained the first and second
histidine boxes in the N-terminal region, but lost the third histidine box. Moreover, the gene
structure of GrFAD2.1 was also different from other FAD2 genes. By using five gene-specific
primers, GrFAD2.1 was not found to be expressed in this study. These observations suggested
that GrFAD2.1 might not be a functional gene. Considering the adjacent location of GrFAD2.1
to GrFAD2.2 on chromosome 13, it could be deduced that GrFAD2.1 might have undergone
significant functional divergence or even have become a pseudogene after originating from an
ancient tandem duplication event of the FAD2 gene in the G. raimondii genome.
Gene duplications play a significant role in the expansion of gene families in the genome
[65,66]. The total number of membrane-bound FAD genes identified in G. raimondii was
much greater than that in Arabidopsis and rice. Phylogenetic analysis of membrane-bound
desaturases in G. raimondii, Arabidopsis and rice indicated that all subfamilies except for the
First Desaturase subfamily contained more gene members in G. raimondii than in Arabidopsis
and rice. The increased number of members of the G. raimondii membrane-bound FAD gene
family belonging to the three subfamilies suggested that they might have undergone
speciesspecific expansion during the process of evolution. In this study, the gene duplication events,
including tandem and segmental duplications, were investigated to elucidate the expansion
mechanism of the membrane-bound FAD gene family in G. raimondii. Four duplicated gene
pairs, including eight genes out of the 19 membrane-bound FAD genes, were identified.
Among them, one tandem duplicated gene pair, GrFAD2.3/GrFAD2.4, belonged to FAD2
cluster of the Omega Desaturase subfamily. A segmental duplicated gene pair, GrDSD1/GrDSD2,
belonged to the Sphingolipid Desaturase subfamily. And the remaining two segmental
duplicated gene pairs, GrSLD1/GrSLD2 and GrSLD4/GrSLD5, belonged to the Front-end Desaturase
subfamily. These results showed that the tandem duplication might contribute to the increasing
size of the FAD2 cluster and that the expansion of the Sphingolipid and Front-end Desaturase
subfamilies was due to the segmental duplication.
Duplicated genes may experience three outcomes, i.e., non-functionalization (loss of
original functions), neo-functionalization (acquisition of novel functions), or sub-functionalization
(partition of original functions), during the process of evolution . Gene expression profiles
can provide useful clues for understanding the function of these genes. According to the
experiments characterizing tissue-specific expression or response to low temperature performed in
this paper, the expression patterns of members in the four duplicated gene pairs were
significantly diverse, which indicated that the functions of the duplicated genes were strongly
differentiated after duplications. The fate of the duplicated genes could be described as
Low temperature is one of the serious environmental stresses affecting cotton development
and production. Previous studies revealed that FAD2 and FAD7/8 genes participated in cotton
adaptation to cold stress [21,25], and accumulating evidence has indicated that many members
of the membrane-bound FAD gene family were involved in the response to cold stress in
numerous plant species [9,10,11,14,15,18,20]. In this study, the expression patterns of the G.
raimondii membrane-bound FAD genes in leaves under low temperature were investigated at the
whole family level. As a result, GrFAD8.1, GrFAD2.2, GrFAD8.2, GrSLD2, GrSLD4, GrDSD1
and GrSLD5 were found to be significantly up-regulated in response to cold stress, which
suggested that these genes might be required to maintain appropriate levels of related unsaturated
fatty acids in cotton plants under low temperature conditions. Conversely, GrFAD5, GrFAD7,
GrFAD2.3, GrSLD1 and GrSLD3 were heavily down-regulated after long periods of cold stress
treatment. In the previous study, two FAD2 genes, designated FAD2-3 and FAD2-4, were
found to be induced in upland cotton (G. hirsutum) under cold stress . In this study, four
functional isoforms of FAD2 genes were identified in G. raimondii, of which GrFAD2.2 was
induced under low temperature, GrFAD2.4 and GrFAD2.5 were slightly up-regulated in response
to cold, and GrFAD2.3 was suppressed under low temperature. These results suggested that
specific isoforms of FAD2 genes might play a vital role in cotton response to cold stress.
SLD genes encoding the sphingolipid 8 desaturases have been well studied in Arabidopsis,
and deficiency of the genes resulted in enhanced sensitivity to prolonged low-temperature
exposure . Here, GrSLD2, GrSLD4, and GrSLD5 were highly expressed after long periods of
cold treatment, whereas GrSLD1 and GrSLD3 were suppressed under low temperatures,
suggesting that SLD genes are critical for cotton response to cold stress.
S1 Fig. Sequence logos of the three histidine boxes in four subfamilies. The height of the
letter designating the amino acid residue at each position represents the degree of conservation.
The numbers on the x-axis represent the residue positions within the boxes. The y-axis
represents the information content measured in bits. Note that all protein sequences in each
subfamily were included in the analysis, with the exception of GrFAD2.1, which was excluded from
the Omega Desaturase subfamily.
Conceived and designed the experiments: SZ JC. Performed the experiments: W. Liu W. Li
QH. Analyzed the data: W. Liu MKD. Contributed reagents/materials/analysis tools: SZ.
Wrote the paper: W. Liu W. Li QH MKD JC SZ.
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