Introduction
Floral scent, which is a mixture
of low molecular weight volatile compounds, is a key factor considered in the
use of garden plants due to the aesthetic and economic value (Giusto et al.
2010). In addition to attracting insects for pollination, the compounds of
floral scent are important for fragrance and medicinal products (Zhu et al.
2016). A mass of volatile compounds were recently identified, and a number of
biosynthesis-related genes were cloned in plants (Lavid
et al. 2002; Shalit et al. 2003; Verdonk et al. 2003; Wu et al. 2004; Boatright et al. 2004; Dexter et al. 2007).
Linalool is an acyclic monoterpene found in the floral scents of many plants. As
an important fragrance material, linalool is widely used in cosmetic products
such as perfumes and shampoos, and in non-cosmetic products like household
cleaners and detergents (Karuppiah et al.
2017). The annual worldwide use of linalool exceeds 1000 tons, which is mostly
extracted from plants (Bickers et al. 2003). Linalool synthase (LIS) is
the enzyme that catalyzes the formation of linalool using geranyl
pyrophosphate as a substrate. LIS has two conserved regions, the aspartate-rich
(DDxxD) motif and the NSE (RLxxDxxxxxxExxxG)
motif, which are essential for binding Mg2+ ions (Karuppiah et al. 2017). In plants, the LIS
gene is firstly cloned from the flower of Clarkia
breweri (Pichersky et
al. 1995). Until now, LISs have
been widely isolated from the species such as Arabidopsis, cotton, papaya, grape, and citrus (Shimada et al.
2014; Zhu et al. 2014; Boachon et al.
2015; Gomes et al. 2016; Huang et al. 2018).
Sweet osmanthus
(Osmanthus fragrans Lour.) is
an evergreen shrub species belonging to the Oleaceae family. As its unique
fragrance and cultural values, sweet osmanthus is a
well-known flower in East Asia, especially in China. Based on their flowering
season and corolla coloration, sweet osmanthus can be
classified into four groups: Yingui, Jingui, Dangui, and Sijigui (Han et al. 2019). The fragrance of the osmanthus flower contains more than 30 chemical substances,
and several related genes have been isolated in the past decade (Wang et al.
2009). Baldermann et al. (2010) cloned a
carotenoid cleavage dioxygenase 1 encoding gene, and
showed its relationship to carotenoid accumulation and volatile emission during
floral development. Han et al. (2016) characterized an OfWRKY3 positively regulating the carotenoid cleavage dioxygenase gene OfCCD4. Xu et al. (2016) identified 10
2-C-methyl-d-erythritol 4-phosphate (MEP) pathway-related genes in O. fragrans,
and OfDXS1,
OfDXS2,
and OfHDR1
expression patterns have a clear diurnal oscillation. O. fragrans var. Jinyu
Taige is a cultivar of the Sijigui
group that has a large number of flowers and a long flowering period (Qin et
al. 2012). Based on the results of previous transcriptome
sequencing of O. fragrans
var. Jinyu Taige at
different stages (Xian et al. 2019), we isolated the OfLIS gene using rapid amplification of cDNA
ends (RACE) technology in this study. These results provide a theoretical basis
for research on the mechanism of aroma formation, the selection of new species,
and improvement of the processing and use of O. fragrans
products, which will support the industrial development of O. fragrans and accelerate the structural reform of the
O. fragrans supply industry.
Materials and Methods
Osmanthus fragrans Lour. var. Jinyu
Taige was planted in 2010 in the garden of the germplasm resource for woody flowers at the Horticulture
Research Institute, Modern Agricultural Science and Technology Innovation
Demonstration Park, Sichuan Academy of Agricultural Sciences, China. Flowers (3
g) from 3 different plants with the same growth status and health at early
flowering stage (EFS), peak-begin flowering stage (PBFS), peak flowering stage
(PFS), and peak-end flowering stage (PEFS) were collected status from September
to October 2017 and quickly frozen with liquid nitrogen for storage at –80°C.
The roots and leaves were rinsed with ddH2O (double distilled H2O),
cleaned with aseptic paper and then stored at –80°C.
Extraction of total RNA and synthesis
of cDNA
According to the instructions of
the RNA prep Pure Plant Kit (DP437, Tiangen Biotech
Co., Ltd., Beijing, China), total RNA was extracted
from O. fragrans
var. Jinyu Taige. Then,
the purity and integrity of the extracted total RNA were examined by spectrophotometry
(UV757CRT, INESA Analytical Instrument Co., Ltd., Shanghai, China) and 1% agarose gel electrophoresis (DYCP-32A, Liuyi
Biotechnology Co., Ltd., Beijing, China), respectively. Finally, according to
the instructions of the Revert Aid First Strand cDNA
Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA), 1.5 µg of
total RNA was used for the synthesis of first-strand cDNA.
Cloning of full-length cDNA of the OfLIS gene
Based on an analysis of transcriptome data (Xian et al. 2019), c162522_g1 in
O. fragrans with a length of 1463 bp was found to have certain homology with the LIS proteins in other plants. To
obtain the full-length sequence of the gene, the following primers were used
for amplification: 5’-RACE-F: AAG CAG TGG TAT CAA CGC AGA GT, 5’-RACE-R: CAA
GTG TTT TCT CGA TCT CTT, 3’-RACE-F: ATC CGA GCT TAA CAA ATG AAA GGC, and
3’-RACE-R: TAC GTT TTT TTTTTT TT. Then, the amplified fragments were recovered
with an agarose gel DNA recovery kit (DP 209,Tiangen
Biotech Co., Ltd., Beijing, China) and ligated into the pEASY-Blunt
Simple Cloning Kit (CB111-01, Trans Gen Biotech Co., Ltd., Beijing, China)
cloning vector and used to transform Escherichia coli strain DH5α.
Three clones cultured overnight at 37°C were selected for sequence
determination by Gen Script Co., Ltd. (Nanjing, China). The sequencing results
were spliced with the c152118_g1 sequence with DNAMAN to obtain the complete
full-length OfLIS cDNA.
Bioinformatics analysis of the OfLIS gene
The open reading frame (ORF) of
the OfLIS
gene was predicted using ORF Finder software (http://www.ncbi.nlm.nih.gov/gorf/gorf.html,
NCBI, Bethesda, Maryland, USA). The derived protein molecular weight and
isoelectric point of OfLIS
were predicted using Prot Param
software (http://web.expasy.org/protparam, SIB Swiss Institute of
Bioinformatics, Switzerland). The three-dimensional homologous modelling of OfLIS was
performed by SWISS-MODEL (http://swissmodel.expasy.org/, SIB Swiss Institute of
Bioinformatics, Switzerland). The phosphorylation sites in the protein sequence
derived from OfLIS
were predicted using Kinase Phos software (http://kinasephos.mbc.nctu.edu.tw/index.php,
Bid Lab, Institute of Bioinformatics, National Chiao
Tung University, Taiwan). OfLIS was compared
with FrLIS
(KX452731), OsLIS
(AK110925), Lllis
(ABD77417), AtLS1(AAO85533),
AaLS1 (GQ338153), CuSTS3-1 (AB857230), PhLS (FJ644546), AmNES/LIS (EF433761), CbLIS (U58314), VvRiLinNer
(JQ062931), GhTPS12 (KJ957818), PaLIS (AAL24105),
CsLIS/NES (KF006849), and OfLis (FJ645727) sequences using
DNAMAN 5.22 software (Lynnon Biosoft,
San Ramon, CA, USA). The Multiple Sequence Alignment website (https://www.ebi.ac.uk/Tools/msa/,
Agilent, Santa Clara, CA, USA) was used for LIS protein phylogenetic tree
construction with 1000 bootstrap replicates. LIS sequence information from
different species was obtained from the National Center for Biotechnology
Information (NCBI).
Expression pattern analysis of the OfLIS gene
The following qPCR (quantitative real-time PCR) primers for the OfLIS gene were designed using Primer Premier
5.0 software (Primer Biosoft International, San
Francisco, CA, USA): OfLIS-qF
(TTC TGA TGG ATG GAT T) and OfLIS-qR (AAG GTC TGG ACG AGT G). The primer sequences used
to amplify OfActin,
an internal reference gene, were OfActin-qF (CAA GAA GAC CAC CAT GCC AAA) and OfActin-qR (AAA
GCT CAC TGC TCA AAC AAC). The qPCR was performed on a
CFX96 real-time quantitative PCR system (Bio-Rad, Hercules, CA, USA) following
the SYBR® Premix Ex Taq instructions (TAKARA Bio,
Otsu, Shiga-ken, Japan) with an annealing temperature of 58°C. The relative
expression level of the OfLIS
gene was calculated using the2−△△Ct method.
Data analysis
Data were analyzed using S.P.S.S.
19.0 software (IBM, Armonk, NY, USA) by Duncan’s multiple range test. The
capital or lower-case letters represented P
< 0.01 or P < 0.05,
respectively. Excel 2013 (Microsoft, Redmond, Washington, USA) was used for
mapping.
Results
Cloning of the OfLIS gene from O. fragrans
The 5'-RACE and 3'-RACE
amplification products were 987 and 862 bp in length,
respectively. After spliced with c162522_g1, the 2911 bp
full-length OfLIS gene sequence (Gen Bank
No.MK563985) was obtained. The sequence encodes an acidic protein composed of
840 amino acid (aa) residues
with a molecular weight of 96.2 kDa and a theoretical
isoelectric point of 5.93. The 5' and 3' untranslated
regions of the gene are 126 and 262 bp in length,
respectively, whereas the ORF is 2523 bp (Fig. 1). Transmembrane domain analysis indicated that 120–150 aa of the N-terminus comprised transmembrane domain (Fig. 2).
Fig. 1: Full length cDNA and deduced amino acid
sequences of OfLIS. The predicted amino acid sequence is listed below
the nucleotide sequence. Note: *, stop codon
Fig. 2: Transmembrane domain analysis of OfLIS
Sequence alignment of OfLIS with other
homologous plant protein sequences
The OfLIS from O. fragrans var. Jinyu
Taige and the related protein sequences from Oryza sativa, Arabidopsis thaliana, Carica papaya, Clarkia breweri,
and O. fragrans var. thunbergii
were aligned using DNAMAN software ((Lynnon Biosoft, San Ramon, CA, USA). The sequence homologies
between OfLIS and PaLIS,
CbLIS, AtLS1, OsLIS
and OfLis are 93, 92, 52, 63 and 53%,
respectively. Conserved domain analysis clearly showed that all proteins
contained the conserved DDxxD and RLxxDxxxxxxExxxG
motifs (Fig. 3). To analyze the phylogenetic relationship between OfLIS and other homologous proteins, a phylogenetic tree
was constructed among 17 LIS members from XXX plant species using the neighbor
joining method. The phylogenetic analysis showed that all members are divided
into monocot group and dicot group (Fig. 4). The members of the dicot group
were further grouped into subfamily I and subfamily II (Fig. 4). OfLIS belongs to subfamily II and has the closest
relationship with PaLIS.
Expression pattern analysis of OfLIS
Tissue expression analysis
showed the OfLIS
expression level is very low in roots and leaves, but is extremely high in
flowers (Fig. 5A). Different stages of flowering expression pattern indicated
that the expression level of OfLIS gradually increases over the four flower stages and
peaks at PFS but then declines at PEFS (Fig. 5B).
OfLIS tertiary structure model construction and
potential phosphorylation sites prediction
The tertiary structure of the OfLIS was predicted using the online software SWISS-MODEL
(Fig. 6). We found that the protein mainly consisted of a helices and random coils. Potential phosphorylation
sites prediction identified 17 potential phosphorylation sites (Table 1), and
531Y is located in the DDxxD motif (Fig. 6).
Discussion
Linalool is one of the main
components of plant floral scents and is widely used in the cosmetic industry
(Jiang et al. 2015; Huang and Hou 2017).
Recently, linalool was found to have unique effects on human health, such as
hypnotic (Linck et al. 2010), anti-inflammatory
(Huo et al. 2013), analgesic (Kuwahata et al. 2013), anti-tumor (Chang and Shen 2014), and anti-anxiety (Cheng et al. 2015)
effects. LIS is a key enzyme in
the linalool synthesis pathway that directly catalyses
the formation of linalool from GPP (L-galactose-1-phosphate phosphatase) (Nagegowda et al. 2008). Deng et al. (2016)
co-expressed AaLS1 with FPPS (farnesyl pyrophosphate
synthase) in Saccharomyces cerevisiae, and
produced 240 μg/L
(S)-linalool. Over-expression of a linalool synthase GhTPS12 in tobacco
can significantly increase the content of linaloolin
comparison to mock (Huang et al. 2018). In Lavandula officinalis, the result of Southernblotting suggests LIS has two copies (Zhang 2006). As one of the top 10 traditional
flowers in China, O. fragrans
has a pleasant fragrance (Zheng et al. 2017).
Tang et al. (2009) isolated an OfLis with 576 aa in O.
fragrans var. thunbergiiin.
In this study, a OfLIS gene encoding 840 aa was obtained in O.
fragrans var. Jinyu Taige. Despite the differences in length, the two proteins
both have a core conserved aspartate-rich motif and an NSE motif, suggesting
the diversification of LIS in O. fragrans.
Phosphorylation is a kind of protein post-translational modification that
plays an important role in the regulation of protein activity (Eberhardt et al. 2012; Hess and Stamler
2012). The protein phosphorylation is manipulated by adding phosphate groups
through protein kinases (MAPK, CDPK, etc.)
and removing phosphate groups through phosphatases (PP1, PP2A, etc.) (Xu and Zhang 2015; Hou et al.
2016; Tiffany and Boudsocq 2019; Chao et al. 2020). In winter
wheat, Xu et al. (2019) perform phosphoproteomic analyses on the different flowering stages
and identify 124 differentially expressed phosphorylated proteins that
participate in translation, transcription, and metabolic processing.
Additionally, there are studies show that phosphorylation is involved in the
regulation of plant aroma biosynthesis (Fallon and Trewavas
1993). 3-hydroxy-3-methylglutary CoA reductase (HMGR)
is a key regulatory enzyme that controls the synthesis of isopentenyl
diphosphate (IPP), an aromatic precursor. In
Arabidopsis, the 577 site of AtHMGR1 was
found to be phosphorylated by BoHRK from
Brassica oleracea (Dale et al. 1995).
In the present study, 17 potential phosphorylation sites were detected in OfLIS, of
which we found that 531Y is located in the conserved DDxxD
domain. Considering the DDxxD domain is the crucial
catalytic site for binding Mg2+ ions, we deduced the phosphorylation
of 531Y may regulate the catalytic activity of OfLIS.
Table 1: Putative phosphorylation sites determined by OfLIS
analysis
Amino acid position |
Amino acid types |
6 |
Serine |
34 |
Tyrosine |
181 |
Tyrosine |
189 |
Tyrosine |
217 |
Serine |
223 |
Tyrosine |
306 |
Serine |
371 |
Tyrosine |
461 |
Tyrosine |
504 |
Serine |
531 |
Tyrosine |
536 |
Serine |
611 |
Tyrosine |
618 |
Tyrosine |
653 |
Tyrosine |
703 |
Tyrosine |
767 |
Tyrosine |
Fig. 3: Alignment of the OfLIS amino acid sequence with LIS from other five plants.
Gene accession numbers: OfLIS, MK563985 for Osmanthus fragrans var.
Jinyu Taige; PaLIS, AAL24105 for Carica papaya; CbLIS, U58314 for Clarkia
breweri; AtLS, AAO85533
for Arabidopsis thaliana; OsLIS, AK110925 for Oryza sativa; OfLis, FJ645727 for Osmanthus. fragrans var. thunbergiiin. The shading color from black to white
represents homology level: 100%, 75%, 50%, and 0%. The conserved motifs are
underlined in black.
Fig. 4: Phylogenetic analysis of LIS
proteins. The bar represents the evolutionary distance. Gene accession numbers:
OfLIS, MK563985 for Osmanthus fragrans var. Jinyu
Taige; PaLIS, AAL24105 for Carica papaya; CbLIS,
U58314 for Clarkia breweri;
AtLS, AAO85533 for Arabidopsis thaliana; OsLIS, AK110925 for
Oryza sativa; AaLS1, GQ338153 for Actinidia arguta; AmNES/LIS, EF433761 for Antirrhinum
majus; GhTPS12, KJ957818 for Gossypium hirsutum; CsLIS/NES,
KF006849 for Camellia sinensis;
VvRiLinNer, JQ062931 for Vitis vinifera; FrLIS,
KX452731 for Freesia hybrida;
MsLIS, AAC37366 for Mentha spicata; PhLS,
FJ644546 for Perilla frutescens;
and Lllis, ABD77417 for Lavandula latifolia
Fig. 5: Expression pattern of OfLIS in different tissues (A) and different flowing stages (B) in O. fragrans. Error bars for qRT-PCR showed the standard deviation of three replicates.
The capital letters represent P <
0.01 lower case letters indicate P <
0.05
Fig. 6: Tertiary structure model
construction of OfLIS. The upper box (labeled with
red) is enlarged, with the right panel representing the 531Y located in the DDxxD motif
Conclusion
In this study, we cloned an OfLIS gene from O.
fragrans
var. Jinyu Taige.
Sequence alignment indicated that OfLIS
contains the conserved DDxxD motif and RLxxDxxxxxxExxxG motif. The phylogenetic analysis revealed OfLIS belongs to subfamily II of the dicot group and has
the closest relationship with PaLIS.
Tissue-specific expression analysis showed that OfLIS
is specifically expressed in flowers and its expression is highest at peak
flowering stage (PFS). We found 17 potential phosphorylation sites in OfLIS, and
531Y is located in the DDxxD motif. The cloning and
expression analysis of OfLIS gene helps
further our understanding of the mechanism of floral scent formation in O. fragrans.
Acknowledgements
This work was financially supported by a special fund from Sichuan
Province financial flower breeding (No. 2016CYTS-010), a special fund from
Sichuan Province financial engineering (No. 2016ZYPZ-025), and the National
Natural Science Foundation of China (No. 31670622).
Author Contributions
Rui Chen, Yuanzhi
Pan and Xiaolin Xian conceived and designed the
experiments; Rui Chen and Yuanzhi
Pan performed the experiments; Haiyan Song analyzed
the data; Xiaolin Xian contributed materials; Ju Hu and Rui Chen wrote the
paper.
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