Abstract
Flavonoids are mainly associated with growth, development, and
responses to diverse abiotic stresses in plants. A growing amount of
data have demonstrated the biosynthesis of flavonoids through
multienzyme complexes of which the membrane-bounded cytochrome P450
supergene family shares a crucial part. However, the explicit
regulation mechanism of Cytochrome P450s related to flavonoid
biosynthesis largely remains elusive. In the present study, we reported
the identification of a stress-tolerant flavonoid biosynthetic
CtCYP82G24 gene from Carthamus tinctorius. The transient transformation
of CtCYP82G24 determined the subcellular localization to the cytosol.
Heterologously expressed CtCYP82G24 was effective to catalyze the
substrate-specific conversion, promoting the de novo biosynthesis of
flavonoids in vitro. Furthermore, a qRT-PCR assay and the accumulation
of metabolites demonstrated that the expression of CtCYP82G24 was
effectively induced by Polyethylene glycol stress in transgenic
Arabidopsis. In addition, the overexpression of CtCYP82G24 could also
trigger expression levels of several other flavonoid biosynthetic genes
in transgenic plants. Taken together, our findings suggest that
CtCYP82G24 overexpression plays a decisive regulatory role in
PEG-induced osmotic stress tolerance and alleviates flavonoid
accumulation in transgenic Arabidopsis.
Keywords: cytochrome P450, heterologous expression, transgenic
Arabidopsis, abiotic stress, flavonoid biosynthesis
1. Introduction
During natural flavonoid biosynthesis, chalcone synthase (CHS) utilizes
phenylpropanoids and malonyl-CoA as general substrates to catalyze the
phenylpropanoid pathway in plants. Following other metabolic routes,
these precursor compounds can be converted into six different
subclasses including flavonols, anthocyanins, flavones, and
flavan-3-ols, the last of which includes proanthocyanidins, catechins,
phenolic acids and flavonols [[46]1]. Various groups of multienzyme
complexes are known to catalyze the biochemical reactions of plant
physiological pathways and their adaptation to ever-changing
environments [[47]2,[48]3]. These complexes of enzymes are mainly
located in the cytosol and also found embedded near the inner side of
the endoplasmic reticulum (ER). The ER-bound enzyme complex belongs to
the cytochrome P450 superfamily [[49]4,[50]5]. In most of the plant
species, cytochrome P450 is found abundantly and produces multiple
groups of secondary metabolites through hydroxylation and
monooxygenation reactions. CYP701, a member of cytochrome P450 encoding
ent-kaurene oxidase, is essential for the biosynthesis of gibberellins
[[51]6,[52]7]. Recent studies have reported the characterization of
CYP90A/B and CYP724, CYP93, CYP73A, CYP75A, and CYP81 as cinnamate
4-hydroxylase, flavonoid 3′,5′-hydroxylase, flavonoid 3′-hydroxylase
and flavone synthase II, respectively, to catalyze the oxidative
reactions during brassinosteroid production and flavonoid biosynthesis
in plants [[53]8,[54]9,[55]10]. Furthermore, several other studies have
discovered the pharmacological importance of the cytochrome P450
supergene family by stimulating plant immunity against various
pathogens [[56]11,[57]12]. Recently, scientists have discovered that
anthocyanins could minimize the risk of many chronic diseases like
hypertension [[58]13] dysentery, diarrhoea and cardiovascular diseases
[[59]14,[60]15].
Safflower (Carthamus tinctorius L.) also known as ‘Bastard saffrons’’
is a highly broadleaf plant belongs to the Asteraceae family. Due to
the increasing demand for safflower oilseed, which highly rich in
conjugated linoleic acid, it has attracted the attention of plant
biologists worldwide. Safflower’s oilseed consists of 80% of
octadecadienoic acid, which can regulate the rate of cholesterol and
avert diseases related to cardiovascular channels [[61]16]. Over 5000
types of phenolic compounds exist across the plant kingdom in which
safflower shares a remarkable variety of flavonoids including carthamin
chalcone glycoside, kaempferol glucosides, hydroxy safflor yellow A and
B, and quercetin glucosides. [[62]17,[63]18]. Therefore, safflower is
radically recommended for its medicinal and economic value in mainland
of China and West Asia. Though the diversity of flavonoid biosynthesis
has been reported in Arabidopsis thaliana seeds [[64]19], the mechanism
of stress-responsive cytochrome P450 genes encoding a single or group
of enzymes promoting the flavonoid biosynthesis pathway in Arabidopsis
remains unexplained. As a point of paramount significance, it is
important to focus on studies related to identification and
characterization of putative cytochrome P450 genes promoting the
flavonoid biosynthetic pathway in plants. In the present study, we
report the discovery and characterization of a 1368bp long fragment of
cDNA which encodes a novel CYP450 (CtCYP82G24) from Carthamus
tinctorius using an expressed sequence, homology-based approach
followed by green fluorescent protein tagging and expression analysis
in a transgenic system. In addition, comparative analyses of qRT-PCR
assay and metabolite accumulation were carried out to examine the
mechanism of PEG-induced osmotic stress tolerance in CtCYP82G24
overexpressed plants by upregulating the transcription of multiple
flavonoid pathway genes. Our results provide adequate information and
novel insight to understand the regulatory mechanism of a new osmotic
stress-responsive CYP gene in transgenic Arabidopsis.
2. Materials and Methods
2.1. Plant Materials, Vectors, and Strains
Carthamus tinctorius cultivar, Jihong No.1 seeds were purchased from
Fuyu Seeds Company, China. Wild-type A. thaliana was grown for 5–7
weeks, and the early flowering plants were subjected to floral-dip
transformation. Agrobacterium tumefacien strain EHA105, Escherichia
coli BL21, E. coli DH5α, the prokaryotic expression vector
(PET28a-CtCYP82G24), (the plant over-expression vector
pBASTA-CtCYP82G24), the subcellular localization vector
(CtCYP82G24-pCAMBIA1302-GFP (green fluorescent protein) was
successfully constructed and preserved the 80% glycerol stock at −80 °C
until next use. Restriction enzymes (BamH1, EcoR1, BglII, and Spe1) and
DNA ligase (T4) were purchased from Takara Biotechnology Company
Beijing, China.
2.2. De Novo Transcriptomic Assembly
A total of 454 sequencing libraries were generated from the total RNA
samples of the early and full flowering stages of Carthamus tinctorius
using a GS-FLX sequencer (Roche, Basel, Switzerland). Clean reads were
obtained, were followed by the deletion the raw data including
low-quality reads, adapter reads, hairpin structural reads and shorter
reads (<50 bp), and were then assembled into unigenes using the MIRA
program [[65]20]. The longest transcripts were further selected as
unigenes for functional analysis by identifying their corresponding
nucleotide sequences. The study of functional annotations and pathway
enrichment analysis of the unigenes were investigated using a
non-redundant protein sequence ([66]http://www.ncbi.nlm.nih.gov) and
non-redundant nucleotide sequences ([67]http://www.ncbi.nlm.nih.gov/).
Further annotations were analyzed in SwissProt. Gene ontology (GO)
annotations were carried out using blast2GO
([68]http://www.geneontology.org/) [[69]21]. To determine the
high-level functional enrichment of biosynthetic systems from
molecular-level information, the Kyoto Encyclopedia of Genes and
Genomes pathways analysis was performed using GenMAPP 2.1
([70]http://www.genmapp.org/).
2.3. Characterization and Phylogenetic Analysis
Based on the information above, the multiple sequence alignment of the
candidate CtCYP82G24 was built using the DNAMAN software (Lynnon Corp.,
Quebec, Canada) and MEGA. v5 by implementing auto-reorder tools
[[71]22]. The occurrence of gaps in the alignment results was removed,
followed by a 1000 rapid bootstraps method. The resulted phylogenetic
tree was edited and visualized using MEGA v5. The number of the open
reading frame was calculated using Laser gene SeqMan II Module DNAStar
([72]www.DNAstar.com). The three-dimensional protein model was created
by implementing Protein Homology/analogy Recognition Engine V 2.0
Phyre^2 software [[73]23]. The expected amino acid sequence,
isoelectric point, and molecular weight of CtCYP82G24 were identified
with the help of ExPASy ProtParam software
([74]http://web.expasy.org/protparam/).
The complete coding sequence of CtCYP82G24 was deposited in GenBank
under accession number [75]MK583014. In addition, the conserved motifs
of the CtCYP82G24 protein were screened and analyzed with the help of
the online web tool Multiple EM for Motif Elicitation version 4.8.1
([76]http://meme-suite.org/tools/meme). The following parameter
settings were used: The per sequence occurrence of the single motif was
optimized from 2 to 10 sequences of amino acids, the number of elements
was set to default, the maximum number of motifs was set to 7, and the
optimal pattern width was fixed from 10 to 250 residues. The hidden
model was generated for individual motifs occupying highly conserved
topology among other species. The alignments of the seven conserved
motifs were obtained from 20 different sequences of various dissimilar
plants. Each model of the aligned sequences was screened across the
CtCYP82G24 full-length protein sequence ([77]Table S1). The results
were represented in seven different colours, including green, yellow,
pink, blue, red, grey, and light green, indicating seven conserved
domains. The graphical representation of protein motifs/domains was
created using the online tool EvolView v.2
([78]http://www.evolgenius.info/).
2.4. Extraction and cDNA Cloning of CtCYP82G24
To isolate the full-length cDNA sequence of CtCYP82G24, the flower
petals of the JH1 cultivar was used as a source of mRNA which was
sampled and placed into liquid nitrogen before RNA extraction. The
total RNA content was extracted using RNA Isoplus (Takara Bio Co.,
Beijing, China), and cDNA templates were synthesized using reverse
transcriptase superscript IV (Thermo Fisher Scientific) in a reverse
transcription PCR system. The amplification protocol includes the
following set of gene primers. The forward primer contained the EcoRI
recognition site along with a start codon for translation initiation.
On the other hand, the reverse primer consists of a BamHI restriction
site just after the stop codon.
CYP-24F:5′ATA
[MATH: GAATTC :MATH]
TTAATCATAGAGCTCCGAAGA3′ (62 °C)
CYP-24R:5′AATA
[MATH: GGATCC :MATH]
ATGGCCGACGACTATGGC3′ (58 °C)
The primers were designed according to the Kyoto Encyclopedia of Genes
and Genomes Pathway information. The exact PCR product of CtCYP82G24
was amplified with Pfu DNA polymerase (Takara, Beijing, China) and then
successfully cloned into the pEASY-T1 vector (Takara, Dalian, China),
and the recombinant construct was further transformed into E. coli
(TransT1) competent cells through the heat and shock method. Then, the
gene of interest in the pEASY-T1 vector was sent for sequencing to
observe any base mutation.
2.5. Subcellular Compartmentalization of CtCYP82G24
The estimated subcellular position of CtCYP82G24 was initially observed
using the LocTree3 online portal
([79]https://rostlab.org/services/loctree3/). Then, the full length of
CtCYP82G24 was amplified using the forward primer CtCYP-24FYXZ:
5′AATAACTAGTATCATAGAGCTCCGAAGA3′ containing the restriction site for
BglII enzymes. The reverse primer CtCYP-24RYXZ: 5′
AATAAGATCTATGGCCGACGACTATGGC3′ contained a Spe1 recognition site. The
positive bands of CtCYP82G24 were recovered from 1% agarose gel after
digestion with a 200 uL double digestion system at 37 °C for 4 h. The
restriction digestion pattern of CtCYP82G24 with BglII and Spe1 was
analyzed on 1% agarose gel electrophoresis. After Sanger sequencing,
the 1368 bp fragment of CtCYP82G24 was fused with the linearized
pCAMBIA1302-GFP-CaMV35S vector using T4 ligase. After re-confirmation
by Sanger sequencing, the recombinant vector
(pCAMBIA1302-CtCYP82G24-GFP-CaMV35S) was successfully transformed into
Agrobacterium tumefaciens (EHA105 strain) following the protocol of
freeze and thaw transformation. At the same time, an empty vector of
pCAMBIA1302-GFP-CaMV 35S alone was also transformed into A. tumefaciens
and was used as a control. Transformed colonies were selected on the
Yeast extract peptone agar plates containing 50 mg/L of streptomycin
and 50 mg/L of kanamycin; they were confirmed by the half colony PCR
method. After confirmation by PCR, the single colony was resuspended in
a YEP liquid medium for 24 h. Lastly, the the Agrobacterium culture was
harvested at an OD[600] of 1.0. The infiltration of onion epidermal
layers with the suspension of Agrobacterium was performed for 20–30 min
and then transferred to a dark room for 26 h. Transformants were
selected for the observation of GFP signals in comparison with the
control under a laser confocal microscope.
2.6. Expression and Induction of Recombinant Protein
The full-length cDNA of CtCYP82G24 was cloned into the prokaryotic
expression vector (pET28a^+) using Pfu DNA polymerase (Takara). The
primer pairs included CtCYP28-F (5′TTAATCATAGAGCTCCGAAGA3′) with an
added EcoRI recognition site and CtCYP28-R (5′ATGGCCGACGACTATGGC3′)
with an added BamHI site. The cDNA library was constructed followed by
amplifying the target bands. After sequence confirmation, the double
restriction digestion of CtCYP82G24 and pET28a^+ was performed using
200 uL plasmid digestion systems. The ligation of the CtCYP82G24
digested product into the appropriate EcoRI, and BamHI sites of the
linearized pET28a^+ plasmid were carried out with the help of the T4
ligase. The recombinant construct (Pet28a^+-CtCYP82G24) was inserted
into the competent BL21 (E. coli cells). The transformed colonies were
detected on an LB agar (solid) medium containing 50 mg/L of kanamycin
using half colony PCR methods. Following the protocol described by
[[80]24] with slight modifications, the CtCYP82G24 recombinant protein
was successfully expressed. For this purpose, the transformed BL21
cells containing pET-28a^+-CtCYP82G24 were cultured in 500 mL LB media
incubated with 50 mg/L of kanamycin. The OD of the inoculums were
measured, and when the growth rate reached a maximum of 2 × 10^8
cells/mL (A600 = 1.0), it was supplemented with 0.4 mM Isopropyl β-
d-1-thiogalactopyranoside (IPTG) under an aseptic environment, and the
incubation of the cells was started at 37 °C for 6 h. After incubation
with IPTG, the cells were transferred to large centrifuge tubes
followed by centrifugation at 12,000× g for 10 min at 4 °C. The cells
were collected by removing the supernatants, and the remaining pellet
was resuspended in a PBS buffer followed by centrifugation at 12,000× g
for 10 min at 4 °C. A 100 mL lysis buffer composed of (12.1 g of
Tris-base, 5.84 g of NaCl, and 0.58 g of EDTA) was added to harvest the
cells. Pulses sonication was performed in three intervals for 15 s
until soluble fractions. Bovine serum albumin and N-terminal histidine
tagging were used for CtCYP82G24 protein purification with an Ni-NTA
affinity column. We first washed the affinity column with the help of
equilibrium buffer containing Na2HPO4 and NaCl, (20 mM:200 mM:500 mM),
and then the CtCYP82G24 product was eluted using a binding buffer (20
mM imidazole). Finally, the adsorbed protein content of CtCYP82G24 was
rinsed in the elution buffer, and the imidazole from the binding buffer
was washed out with the help of the equilibrium buffer. After the
thorough purification of Pet28a^+-CtCYP82G24, the soluble products were
separated on 12% SDS-PAGE, and the results were visualized with the
help of Coomassie brilliant blue.
The quantification of metabolites were performed according to the
instructions of [[81]25]. The products were further analyzed using a
high-performance liquid chromatography system equipped with a
multiple-wavelength detector (MD-2010, JASCO, Japan). The
quantification of samples was analyzed by the measurement of the
integrated peak against two different authentic standards, including
rutin and dihydrokaempferol. The data were further analyzed using the
statistical significance of the differences by measuring the retention
time and abundance values of three independent replicates at 415 nm UV
spectra. The column temperature was kept 28 °C with a flow rate equal
to 0.7 mL/min according to the previous protocol of [[82]26].
2.7. Vector Construction and Agrobacterium-Mediated Transformation of
Arabidopsis
The full length of CtCYP82G24 from the flower petals of JH1 cultivar
was amplified using the CYP-24F and CYP-24R pair of primers. The
CtCYP82G24 full-length coding region was ligated into the BamHI/EcoRI
digested pBASTA-harboring BAR gene under the control of the 35S (CaMV)
promoter. T4 ligase was used to ligate the gene of interest and plant
over-expression vector according to [[83]27]. The recombinant product
was transformed into DH5α E.coli-competent cells by the heat and shock
method, and the positive colonies were detected with colony PCR and
confirmed by Sanger sequencing. The recombinant vector
pBASTA-CtCYP82G24, as well as the pBASTA empty vector, was transformed
into A. tumefaciens (EHA105 strain). The genetic transformation of wild
type A. thaliana was carried out using the floral-dip infiltration
method with slight changes according to [[84]28]. T1 seeds were screen
out by spraying BASTA to set T2 and T3 generations of transgenic A.
thaliana. The PCR amplification of the CtCYP82G24 transgene, the
herbicide resistance BAR gene, and the NOS terminator gene were
performed using GoTaq DNA polymerase (Promega Corp, Madison, WI, USA)
and the corresponding primer pairs ([85]Table S2).
2.8. Metabolites Accumulation in Transgenic Plants
We performed different elicitation experiments on transgenic plants
maintained previously in a growth chamber of the Ministry of Education
Engineering Research Center of Bioreactor and Pharmaceutical
Development at Jilin Agricultural University. To investigate the
abundance of metabolite accumulation, the homozygous T3 plants was
selected at (approximately 25 days 12-leaf stage) and then incubated
with PEG-induced osmotic stress (6% PEG6000) at four different time
periods (0, 3, 6, and 9 h) in a Hoagland’s solution. Five individual
transgenic plants were selected to reduce the possible errors obtaining
from the differences of individual plants. The leaves of transgenic T3
homozygous plants were weighted precisely to a 1 g fine powder and then
soaked in 14 mL distilled water–alcoholic solution for the
ultrasonication extraction of the metabolites using the following
conditions: Extraction temperature, 60 °C; extraction period, 30 min
twice; and centrifugation at 5000 rpm for 10 min. An aliquot of 0.5 mL
of sample (1 mg/mL) was mixed with 0.1 mL of 10% aluminum chloride and
0.1 mL of potassium acetate (1 M). In this mixture, 4.3 mL of 80%
methanol was added to make a 5 mL volume. This mixture was vortexed,
and the absorbance was spectrophotometrically measured at 415 nm. The
total flavonoid content was expressed as milligrams of
rutin/dihydrokaempferol equivalent (CE) per 100 g fresh weight or dry
weight. Each sample was quantified using three biological replicates.
2.9. Quantitative RT-PCR Analysis
To further explore the CtCYP82G24 transcript abundance in transgenic
plants, five homozygous T3 lines of A. thaliana fresh leaves were
converted to a fine powder in liquid nitrogen and then stored at −80 °C
until cDNA template synthesis for qRT-PCR analysis. The relative
expression level of CtCYP82G24 in five individual transgenic T3 plants
was analyzed, and the transcript abundance was measured using the
2^-△△Ct method. Furthermore, the expression level of CtCYP82G24 in the
selected transgenic lines under PEG-simulation at different time
periods was also investigated. For this purpose, healthy plants were
individually treated with PEG6000 following four incubation periods
after 0, 3, 6, and 9 h. After PEG induction, the total RNA content was
isolated by using RNAiso plus reagent (Takara, Beijing). The
first-strand cDNA was synthesized through reverse transcription PCR
using the PrimeScript RT reagent kit (Takara, Beijing) according to
manufacturer’s instructions. Simultaneously, under the same conditions,
the qRT-PCR assay was also investigated in ARB3-CYP24 transgenic line
to determine the transcription levels of the eight core structural
genes of the flavonoid pathway compare to wild-type plants. All qPCR
reaction were performed in a fast real-time PCR system (Applied
Biosystems 7300/7500/7500, CA, USA) using 20 μL reaction including 10
μL SYBR Premix Ex Taq (Tli RNaseH Plus), 0.3 μL ROX Reference Dye, 0.4
μL F/R primer, 1 μL template and 7.9 μL ddH[2]O. The thermal cycle
includes an initial step of denaturation at 94 °C for 30 s), followed
by 40 cycles at 94 °C for 30 s and final step at 60 °C for 3 s. All
qRT-PCR reactions were carried out in three replicates, and the
relative expression was normalized with the 18s ribosomal RNA gene
(AT5G38720.1) which was used as a housekeeping gene in our analysis.
The expression level was calculated according to the 2^−△△Ct method.
The detail list of primers used for the qRT-PCR analysis of key
structural genes of the flavonoid pathway in transgenic Arabidopsis is
listed in [86]Table S3.
2.10. Statistical Analysis
The data are demonstrated as mean values ± Standard Deviation. with
three independent biological replicates. The each group differences
between mean values were measured by a one-way analysis of variance
using Statistix 8.1 software (USA). Asterisks indicate statistical
significance (* p < 0.05, ** p < 0.01).
3. Results
3.1. Discovery and Functional Annotation of CtCYP82G24
The RNA-seq transcriptomic data of the Carthamus tinctorius cultivar
Jihong No. 1 were created using a GS-FLX sequencer (Roche, Basel,
Switzerland) and then submitted to NCBI under accession number
PRJNA399628. After deleting the low-quality reads, adapter reads, short
and poly-N reads, a total of 577,664 and 562,930 high qualities reads
from 583,440 and 567,884 raw reads were obtained in the early and full
flowering, respectively. A total of 51,591 transcripts were assembled
using the MIRA program [[87]25]. The average length of the unigenes
ranged within 679–5109 bp. Moreover, these unigenes were further
assigned to Go terms, which were classified into a total of 43
functional groups mainly divided into three Go categories, including
molecular function, cellular components, and biological processes. A
high number of unigenes were assigned to biological processes followed
by cellular and metabolic processes. To categorize the physiological
pathways specific to Carthamus tinctorius, a total of 51,591 unigenes
were annotated and mapped against the Kyoto Encyclopedia of Genes and
Genomes (KEGG) database, resulting in 281 KEGG pathways in which 187
pathways were predicted as up-regulated and 189 as down-regulated
during the metabolic process. Based on predicted open-reading frames,
the unigenes were further subjected to cytochrome P450 database
([88]http://drnelson.uthsc.edu/CytochromeP450.html). The candidate
CYP450 was named according to the shared homology with reference plant
(CtCYP82G24).
3.2. Phylogenetic and Conserved Domain Analysis
To investigate the phylogenetic relationship, a maximum likelihood
phylogenetic tree was created by adding CtCYP82G24 sequences from
different species to the alignment using the 1000 rapid bootstraps
method. The results revealed that the CtCYP82G24 amino acid sequence
shared high similarity with homologous sequences from Cynaracardunculus
var. scolymus (97.7%), Helianthus annuus (95.1%), Artemisia annua
(90.6%), Lactuca sativa (87.3%), Aristolochiadebilis (85.71%),
Arabidopsis thaliana (69.55%) and more than 60% similarity with
homologous sequences from Citrus Clementina, Glycine max, Catharanthus
roseus and Quercus suber ([89]Figure 1A). To identify the conserved
protein motifs of the CtCYP82G24 protein, we screened other
CtCYP82G24-like proteins from different plant species using the MEME
online tool [[90]29]. We identified seven conserved motifs with
different domain positionings ([91]Figure 1B). Five of the conserved
domains were found consistent with the presence of CG-1 and
S-adenosyl-L methionine binding sites. The putative NAD(P)H-bispecific
enzyme and calcium-binding domains were also observed. Altogether, the
distributions of these conserved domains were identified as
stress-responsive elements in Arabidopsis [[92]30] and other plants
[[93]31,[94]32,[95]33], suggesting the importance of CtCYP82G24 to
environmental stress responses.
Figure 1.
[96]Figure 1
[97]Open in a new tab
Phylogeny analysis and identification of the conserved domains (A)
Phylogenetic analysis using the 1000 rapid bootstraps method was
created with the MEGAx online tool. The online tool of the MEME server
was used to identify the conserved motifs. The green, yellow, pink,
blue, red, grey and light green colors represent motifs 1, 2, 3, 4, 5,
6 and 7, respectively. The indication of the grey line length denotes
the sequence length. The presence of each block at multiple positions
determines the location of the conserved motif to its matching one. The
tree and motifs parameters were edited using the EVOLVIEW online tool
(B) Conserved motif compositions and the representation of logos. The
capital letters in the logos of individual motif represent more than
70% ratio of the conserved amino acids. The Arabic numerals appear
beneath uppercase letters and indicate the width of the conserved
motif.
3.3. Cloning and Characterization of CtCYP82G24
The full-length cDNA sequence of CtCYP82G24 was successfully cloned
from Carthamus tinctorius using a gene-specific primer pair ([98]Figure
S1). A 1368 bp long fragment was produced by PCR. This complete cDNA
sequence contains an open reading frame encoding 455 amino acids with a
6.55 theoretical isoelectric point and 51.8 kDa predicted molecular
weight. Further sequential characterization and phylogenetic analysis
revealed the presence of serine/threonine conserved domain at the
N-terminus. This indicates the origin of CtCYP82G24 with the
oxidoreductase superfamily, which could be subject to various kinds of
biochemical regulatory reactions at the cellular level. An analysis of
the 3D protein model revealed certain physiological domains consistent
with other plants. One of the domains contained a loop-helix grasp-like
motif ([99]Figure S2). The instability index (II) was recorded at
36.06.
3.4. Subcellular Localization of CtCYP82G24
The subcellular localization of CtCYP82G24 was computationally
determined using the online the LocTree3 portal. Each prediction was
calculated with a confidence score (0 = unreliable and 100 = reliable)
and gene ontology (GO) term of the predicted localization class. The
result predicted that CtCYP82G24 might be localized to the cytoplasm or
endoplasmic reticulum ([100]Figure S3A). To experimentally determine
the CtCYP82G24 localization, the complete cDNA of CtCYP82G24 was cloned
into the BglII-Spe1 digested site of a pCAMBIA-1302 vector containing a
GFP driven by the cauliflower mosaic virus 35S promoter ([101]Figure
S3B). The recombinant construct (pCAMBIA1302-CtCYP82G24-GFP-CaMV35S)
and empty pCAMBIA-1302 were efficiently transformed into the
Agrobacterium EHA105 strain. As shown in [102]Figure 2, the onion
epidermal cells harboring the fusion construct (pCAMBIA1302-CtCYP82G24)
were visualized under a confocal laser scanning microscope. The
emission of GFP signals was detected primarily in the cytosol. The
fluorescence of the pCAMBIA-1302 empty vector was used as a control.
Our findings indicate the localization of CtCYP82G24 to cytosol,
supporting previous reports on chalcone synthase and chalcone isomerase
localized to cytoplasm and nuclei [[103]34]. Chalcone synthase (CHS)
and chalcone isomerase (CHI) were found to be actively involved during
flavonoid biosynthesis in Arabidopsis. These results provided important
clues to support the hypothesis that CtCYP82G24 is capable of
catalysing cellular-based biological reactions in plants.
Figure 2.
[104]Figure 2
[105]Open in a new tab
Transient transformation of CtCYP82G24. Subcellular localization of the
pBASTA1302-green fluorescent protein (GFP)-CtCYP82G24 in onion
epidermal cells. The fluorescence signals were detected using a
confocal laser scanning microscope.
3.5. Heterologous Expression and In Vitro Enzymatic Activity of CtCYP82G24
To confirm the function of CtCYP82G24 in vitro, we cloned the
CtCYP82G24 cDNA into the expression cassettes of the pET28a^+ vector
([106]Figure S4) and subsequently transformed them into E. coli BL21
DE3 cells using heat and shock transformation. The heterologously
expressed CtCYP82G24 recombinant protein was primarily detected by
Coomassie blue-stained SDS-PAGE. The bands of the purified samples had
molecular weights of approximately 51.8 kDa, which was consistent with
the predicted size of the CtCYP82G24 protein ([107]Figure S4A,B). The
protein extract obtained from pET-28a+ alone was used as a control. The
purification of the CtCYP82G24 protein was obtained using BSA and
affinity chromatography ([108]Figure S5). The in vitro enzymatic
activity of CtCYP82G24 was investigated using rutin and
dihydrokaempferol as substrates. Reaction products were detected by
HPLC. The detection of the solvent peaks and a product P* was observed
when rutin was kept as a substrate. The retention time of the product
P* was approximately consistent with that of the authentic standard of
rutin ([109]Figure 3). However, the other substrate (dihydrokaempferol)
was not catalyzed by CtCYP82G24, which further suggests that CtCYP82G24
may have strict substrate specificity. Additionally, the kinetic assay
of CtCYP82G24 using a range of rutin concentrations showed that the
V[o](reaction velocity) resulted in a rectangular hyperbola bar where
the Vmax value was 29.9 ± 0.14 μM/min and the Km was 5.37 ± 0.123 mM
([110]Figure S6). Our results showed the increased tendency of
CtCYP82G24 corresponding to the biosynthesis of flavonoids by
catalyzing the upstream precursor molecules or core intermediates of
the pathway, thus revealing the partial function of CtCYP82G24.
Figure 3.
[111]Figure 3
[112]Open in a new tab
The chemical structure of rutin and dihydrokaempferol metabolites and
their in vitro enzymatic activity and a high-performance liquid
chromatography (HPLC) profile against the purified recombinant
CtCYP82G24 protein. The protein extract was monitored at 415 nm on an
Agilent Zorbax SB-C 18 column (4.6 × 150 mm, 5 μm) with
methanol:acetonitrile (v:v of 1:10) as mobile phase A and 0.4%
phosphoric acid as phase B. The metabolites were quantified using the
aluminum chloride colorimetric method described by [[113]35]. The total
metabolite content was expressed as milligrams of rutin equivalent (CE)
per 100 g dry weight (DW). (A) Peak labelled dihydrokaempferol and (B)
peak marked as rutin was used as the authentic standards. (C) HPLC
profile of the reaction product P* catalyzed by CtCYP82G24. The
retention times of rutin and product P* showed close proximity in peak
4, which was approximately 6.3 min.
3.6. Identification of CtCYP82G24 Overexpressed Transgenic Lines of
Arabidopsis
Homozygous T3 transgenic seeds of Arabidopsis were grown in the growth
chamber of the Ministry of Education Engineering Research Center of
Bioreactor and Pharmaceutical Development at Jilin Agricultural
University (Jilin, China) until the flowering stage. A selection of the
transgenic plants was performed initially with by spraying (20 ug/mL)
Basta solution. Healthy and viable transgenic plants were further
selected for RNA extraction and cDNA synthesis by RT-PCR. The primer
pair CYP-24F/R was used to amplify the full-length coding regions of
CtCYP82G24 using the cDNA of the transgenic plants as the template.
Five out of six transgenic plants with a high copy number of CtCYP82G24
were detected using 1% agarose gel electrophoresis ([114]Figure 4A).
The further identification of the transgenic plants was conducted by
amplifying the herbicide resistance gene (BAR) to detect the presence
of the pBASTA-CtCYP82G24 transgene ([115]Figure 4B). We also identified
the nopaline synthase (NOS) terminator gene in the selected transgenic
Arabidopsis plants for further confirmation ([116]Figure 4C). The
results confirmed the successful integration of CtCYP82G24 into A.
thaliana. We also performed Southern blot hybridization to detect the
specific exogenous CtCYP82G24 copy number. A single restriction enzyme
(HindIII) digestion of the genomic DNA isolated from the transgenic
plants allowed for the fragmentation of the reporter gene. As described
in [117]Figure 4D, the transformants were detected on 1% agarose gel
after hybridizing with the digoxigenin (DIG)-1-dUTP probe and chemical
labelling with DIG high prime. The products were immobilized by
transferring them onto a nylon membrane that indicated the size of the
expected BAR product together with the flanking sequence probes,
thereby confirming the presence of several copy numbers of the
exogenous CtCYP82G24 within the transgenic Arabidopsis lines.
Figure 4.
[118]Figure 4
[119]Open in a new tab
Detection of transgenic Arabidopsis lines harboring the
pBASTA-CtCYP82G24 transgene. The growth stage for the detection was
selected approximately 25 d at the 12-leaf stage. (A) Detection of
CtCYP82G24 (1368bp) product in transgenic plants using the CYP24R/F
primer pair where M: Marker; P: Plasmid, and lanes 1–6 indicate
different lines of CtCYP82G24—overexpressed transgenic plants. (B) PCR
amplification of the herbicide resistance gene (BAR) used as a
selectable marker for genetic transformation of Arabidopsis. WT
represents the negative control, P is the recombinant vector of
pBASTA-CtCYP82G24 that was used as the positive control, and lanes 1–6
indicate different lines of CtCYP82G24 transformed plants. (C) Positive
PCR detection of the NOS terminator gene in the aforesaid transgenic
lines. P: Plasmid (positive control); WT: Negative control, and lanes
1–6 show the presence of the NOS terminator gene. (D) Southern blot
analysis of transformed Arabidopsis through BAR probe hybridization.
Where the representation was indicated as M: Marker; P: Plasmid—as
positive control; Wt: Wild type—as a negative control, and lanes 1–6
represent transgenic lines.
3.7. Expression Profiling of CtCYP82G24 and Metabolite Accumulation in
Transgenic Plants under PEG-Induced Osmotic Stress
Transgenic homozygous T3 plants were equally subjected to PEG-induction
(6% PEG6000) in Hoagland’s solution for four different durations
including 3, 6, and 9 h, with 0 h as the control. Then, we extracted
the total RNA from the leaves of the five homozygous transgenic T3
Arabidopsis. The template cDNA of the individual plant was amplified
using reverse transcription PCR. Each CtCYP82G24 transgenic plant
demonstrated variable expression levels under various durations of
simulated PEG stress. In addition, we also investigated whether the
transcript levels of CtCYP82G24 corresponded to the metabolite
accumulation in transgenic plants. The qRT-PCR analysis was conducted
at various times of elicitor incubation using the qRT-PCR primers of
CtCYP82G24 with 18S ribosomal RNA as the internal reference gene. The
rate of metabolite aggregation was subsequently measured in the
selected transgenic T3 plants. We used ARB1-CYP24 to ARB5-CYP24 as the
hypothetical code for describing CtCYP82G24-overexpressed lines of
Arabidopsis in our study. Furthermore, ARB1-CYP24, ARB2-CYP24, and
ARB3-CYP24 correspond to lane 1, 2, and 3 of the previous [120]Figure
4; however, because of the presence of transgenes in a low copy number,
we followed the representation of ARB4-CYP24 and ARB5-CYP24 to lane 5
and lane 6, respectively. As shown in [121]Figure 5, the CtCYP82G24
transcript levels in the ARB1-CYP24 line increased at 3 and 6 h after
PEG induction, whereas the accumulation of the flavonoid compounds
increased over time and peaked at 6 h after PEG induction. A further
comparative analysis of CtCYP82G24 expression and flavonoid
accumulation in ARB2-CYP24 line demonstrated a variable pattern under 3
h PEG induction compared to 0 h. Contrary to the transcript abundance
of CtCYP82G24 in ARB3-CYP24, the metabolite biosynthesis was increased
with PEG treatment at 6 h and then decreased after 9 h, suggesting the
early stress-responsive mechanism of CtCYP82G24 compared with 0 h
treatment. Similarly, the result of ARB4-CYP24 and ARB5-CYP24 line
indicated the up-regulation of CtCYP82G24 and peaked at 3 and 6 h, and
metabolite biosynthesis was also increased gradually at these time
points. However, the transcript abundance of CtCYP82G24 and metabolite
aggregation declined at 9 h of PEG treatment in ARB5-CYP24 line. Based
on the relative fold expression of CtCYP82G24 coupled with differential
metabolite plasticity after PEG simulation, we speculate that
CtCYP82G24 transcription is up-regulated during the early phases (3 and
6 h) of PEG stress. Altogether, these results suggested that CtCYP82G24
is an early osmotic stress-responsive gene which could promote
flavonoid biosynthesis in transgenic Arabidopsis.
Figure 5.
[122]Figure 5
[123]Open in a new tab
The PEG-induced expression level of CtCYP82G24 associated with a
notable metabolite accumulation in transgenic Arabidopsis. (A) The
expression level of CtCYP82G24 in transgenic lines (B) PEG-induced qRT-
PCR assay and quantification of metabolite accumulation in
CtCYP82G24-overexpressed transgenic plants at four different periods.
Asterisks indicate statistical significance (* p < 0.05, ** p < 0.01).
The relative expression level of CtCYP82G24 was compared with that of
the 18s ribosomal RNA gene (internal control). The data were calculated
using the 2^−△△Ct method. (C) ARB3-24 phenotypes under PEG stress at 0,
3, 6, and 9 h intervals. The growth stage for expression and metabolite
analysis was selected approximately 25 days old at 12-leaf stage.
3.8. Overexpression of CtCYP82G24 Induced the Expression of Flavonoid Pathway
Genes in Transgenic Arabidopsis
The influence of CtCYP82G24 overexpression on the downstream regulation
of key structural genes of the flavonoid biosynthetic pathway in
transgenic Arabidopsis under PEG simulation was analyzed by a
quantitative real-time PCR (qRT-PCR) assay. Our previous results
demonstrated that CtCYP82G24 was most abundantly expressed in the
ARB1-CYP24, followed by ARB3-CYP24 and ARB5-CYP24 overexpression lines
([124]Figure 5). Even so, the expression level of CtCYP82G24 was higher
in the ARB1 line, and the ARB3 line showed a relatively high quality
and the strongest phenotype compared to ARB1. We therefore chose the
ARB3 line to investigate further insights into the role of CtCYP82G24
in flavonoid biosynthesis and its contribution to the enhancement of
the osmoregulatory mechanism. The result of the qRT-PCR assays
indicated that the expression level of most of the flavonoid pathway
genes, including PAL (Phenylalanine ammonia lyase), CHI (Chalcone
isomerase), CYP82G1 (Cytochrome P450 monooxygenase), F3′H (flavonoid
3′-hydroxylase), and FLS (Flavonol synthase) were more abundantly
up-regulated in the ARB3-CtCYP82G24 overexpression line than that of
wild-type plant ([125]Figure 6).
Figure 6.
[126]Figure 6
[127]Open in a new tab
Downstream regulation of key structural genes of the flavonoid
biosynthetic pathway in wild-type and CtCYP82G24 overexpressed
transgenic line. The blue colour bars represent the expression level of
flavonoid biosynthetic genes in WT plants under PEG-induced osmotic
stress at different time points. The red colour bars indicate
quantitative RT-PCR assays of eight core flavonoid pathway genes in
ARB3-transgenic under the same treatments of PEG stress. The expression
levels of each transcript were expressed using the 2^−△△Ct method as
compared to the control plants. Asterisks indicate statistical
significance (* p < 0.05, ** p < 0.01). The genes were: PAL
(Phenylalanine ammonia lyase), F3′5′H (Flavonoid 3′,5′-hydroxylase),
DFR (Dihydroflavonol 4-reductase), CHI (Chalcone isomerase), CYP82G1
(Cytochrome P450 monooxygenase), F3′H (flavonoid 3′-hydroxylase), ANS
(Anthocyanidin synthase) and FLS (Flavonol synthase).
Nevertheless, the expression of F3′5′H (Flavonoid 3′,5′-hydroxylase),
DFR (Dihydroflavonol 4-reductase), and ANS (Anthocyanidin synthase)
were significantly lower than that of wild type plants. Those results
may indicate that the mediation of CtCYP82G24 is involved in the
flavonoid pathway by regulating the expression of flavonoid-associated
genes in transgenic plants under PEG-induced stress. Similarly, the
biosynthetic mechanism of the different groups of flavonoid in plants
is strongly regulated by the spatial and ectopic expression of these
genes, which can influence the metabolite profiling of these compounds
[[128]36,[129]37]. Our findings also suggest that the overexpression of
CtCYP82G24 could maintain intracellular osmotic balance and minimize
the risk of cell membrane damage, which contributes to osmotic
adjustment in transgenic Arabidopsis. In addition, the transcript
levels of ANS and FLS showed no significant difference than the 0 h
treated plants and carried the lowest expression levels under PEG
induction. In agreement with our results, several copies of FLS and ANS
in various plants including Arabidopsis have been reported that are
differentially expressed in a tissue-specific manner, which controls
the amount and type of flavonoid compounds
[[130]1,[131]38,[132]39,[133]40,[134]41]. Concludingly, our findings
suggested that the overexpression of CtCYP82G24 in transgenic
Arabidopsis strongly enhances PEG-induced osmotic stress responses by
regulating the transcription of key flavonoid pathway genes.
4. Discussion
Flowering is considered to be the most critical component in
developmental processes because it ensures the growth, survival, and
reproduction of flowering plants [[135]42]. The genes in C. tinctorius
that were expressed during four flowering stages were found to be
mostly associated with flavonoid biosynthesis [[136]43,[137]44]. Under
various stress conditions, the biosynthesis of organic compounds
stimulated the defense system of plants and triggered conformational
changes in biological structures that could efficiently influence the
immune system of plant cells [[138]45,[139]46,[140]47]. The CYP450
supergene family has been widely investigated in many plants. Members
of this family catalyze phytochemical and biosynthetic reactions that
produce metabolites such as phenols, glucosinolates, phytohormones,
signaling compounds, terpenoids, and flavonoids. CYP450s also play
essential roles in regulating the homeostasis of secondary plant
metabolism and hormonal crosstalk between signaling molecules
[[141]11,[142]12]. The pharmacokinetic capacity of the CYP450
superfamily in association with the accumulation of metabolites has
been reported previously [[143]6,[144]7,[145]48]. However, to date,
there is no such report available on the C. tinctorius CYP450 gene
involved in osmotic stress tolerance capability by activating
biosynthesis of flavonoids in transgenic Arabidopsis. Based on a
comprehensive RNA-seq data analysis and gene annotation investigations,
we aimed to assembled gene families of Jihong No.1 (JH1) cultivar of C.
tinctorius that participate in flavonoid metabolism (data not shown). A
candidate CtCYP82G24 (CYP450-gene) was figured out in the KEGG database
and followed by various in silico identifications ([146]Figure 1) to
speculate the importance of CtCYP82G24 during flavonoid biosynthesis.
In this study, we preliminarily identified and characterized the
partial function of a stress responsive CtCYP82G24 under the control of
35S promoter ([147]Figure S7) underlying the molecular mechanism of
enhanced flavonoid biosynthesis in transgenic Arabidopsis.
The subcellular studies of biosynthetic enzymes related to flavonoid
biosynthesis at the cellular level have been well studied. Most were
localized in cytosol and nucleus [[148]49,[149]50], and very few of
them were localized in the vacuole [[150]51,[151]52] and endoplasmic
reticulum [[152]53]. Another group of enzymes was likely expressed in
chloroplast and cytomembrane [[153]54]. Flavonoid biosynthetic enzymes
that are located next to plant nuclei and other cellular compartments
provide DNA protectant properties against oxidative stress and UV
radiation. The conversion of these genes into proteins and enzymes
following isomerization and hydroxylation explained the regulation of
crucial biological pathways during plant development [[154]55,[155]56].
Our findings implied that CtCYP82G24 was localized in the cytosol using
the pCAMBIA1302-GFP fusion construct in onion cells, which is
consistent with previous studies which also reported the localization
of several other genes promoting flavonoid biosynthetic pathways in
cytomembranes and nucleus [[156]50,[157]54]. Based on these results, we
demonstrated the potential role of CtCYP82G24 in promoting flavonoid
accumulation in Arabidopsis. However, the details of the regulatory
mechanisms of flavonoid metabolism such as location, transport, and
subsequent trafficking to numerous cellular compartments remain
unclear. There seems to be a close relationship among bio-structure,
transportation, and deposition of cells from flavonoids. Therefore, to
fully understand the regulatory mechanisms of flavonoids, it is
necessary to consider not only their metabolism but also the
transportation and storage of the final product.
Combinational biosynthesis approaches such as E. coli and Saccharomyces
cerevisiae host systems are widely used to produce various bioactive
compounds by providing an artificial biosynthetic pathway of the gene
cluster construct of an organism [[158]57,[159]58,[160]59]. Different
substrates that were initiated from tyrosine and phenylalanine amino
acids were efficiently coexpressed using heterologous systems that
could catalyze compounds such as curcumin, resveratrol, naringenin, and
genistein. Additionally, E. coli cells incubated with precursor
molecules such as carboxylic acids have produced unknown flavonoids
[[161]60]. We also investigated the in vitro enzymatic activity of
CtCYP82G24 by incorporating the PET28a-CtCYP82G24 vector construct into
E. coli BL21 (DE3). To confirm the in vitro function of CtCYP82G24, the
purified protein was incubated with two different substrates—rutin, and
dihydrokaempferol. The reaction products, which were analyzed by
high-performance liquid chromatography ([162]Figure 3), showed that
CtCYP82G24 was effective in catalyzing rutin but not dihydrokaempferol.
These findings are compatible with the possibility of the
CtCYP82G24-induced de novo biosynthesis of flavonoids in the host
system. In addition, one relatively well-known characteristic of the
computational prediction of metabolite biosynthesis is to recognize
metabolically reactive atomic positions in a molecule, also called the
sites of metabolism (SoMs) [[163]61]. The importance of the prediction
of SoMs is crucial to know the position of a labile atom and to predict
the chemical structure of metabolite where a biochemical reaction is
likely to occur. In the present study, we also predicted the
computational structure of rutin against a comprehensive metabolite
prediction tool (GLORY) that contained the cytochrome P450 prediction
module according to the instructions of [[164]62]. The rutin metabolite
structure prediction revealed that C26 is a potential site of for
CYP450-induced hydroxylation ([165]Figure S8).
Furthermore, to emphasize the importance of CtCYP82G24 in plant stress
responses, the transcript abundance and its reciprocal relationship
with the accumulation of metabolites in the transgenic Arabidopsis
under PEG-induced osmotic stress at different time points were also
determined ([166]Figure 5). The CtCYP82G24 mRNA transcript in the
ARB1-CYP24 line notably increased after 3 and 6 h of PEG induction,
whereas the accumulation of the flavonoid compounds increased over time
and peaked at 6 h after PEG induction when compared with 0 h.
Similarly, the expression level of CtCYP82G24 and flavonoid
accumulation in ARB2-CYP24 line slightly increased at the 3 h time
point compared to the 0 h control plants. Furthermore, the expression
level of CtCYP82G24 in ARB3-CYP24 and ARB4-CYP24 showed that an opposed
pattern of metabolite profile was initially increased with PEG
treatment at 6 h and then decreased after 9 h. These results indicate
that statistically significant differences occurred at different times
after PEG induction. We found that PEG pretreatment induced the
up-regulation of CtCYP82G24 up to two-to-three fold through the
flavonoid dependent pathway in transgenic Arabidopsis. Those findings
may also suggest that CtCYP82G24 is involved in the flavonoid pathway
by regulating the transcription of the core flavonoid
pathway-associated genes under PEG-induced osmotic stress ([167]Figure
6).
In a further analysis, we detected an increase antioxidant activity of
CtCYP82G24 during the in vitro investigation on the presence of free
radicals in CtCYP82G24 transgenic Arabidopsis (data not shown). The
deposition of flavonoids is not limited to vacuoles and cell walls, as
it also occurs in chloroplasts [[168]63,[169]64]. Flavonoid compounds
associated with antioxidant properties have been mostly reported as the
core inhibitors during auxin transport in plants [[170]65,[171]66]. The
ability of flavonoid molecules to modulate the movement of auxin and
several other cellular functions in plants [[172]67] is because of the
affinity of flavonoids to attach with the ATP binding sites of various
proteins [[173]68]. Moreover, a general stress response may cause the
downregulation of photosynthetic reactions by closing the stomatal
aperture in plants and turn on other unusual energy resources as an
alternate mechanism [[174]69,[175]70,[176]71]. Based on these reports,
the concept of flavonoid-associated osmotic stress signaling originates
with conceivably active antioxidant properties. Our results also
speculate that the differential expression of several constitutive
genes in PEG-responsive overexpression lines of Arabidopsis reveals
other signaling systems including stomatal closure/aperture. Our
results are in agreement with the findings of [[177]72], who found that
over-expression of the flavonol synthase gene (TaFLS) in wheat
stimulates antioxidant activity by inducing flavonol biosynthesis, thus
promoting the enlargement of the stomatal aperture. From here on, we
also suggest that there may be an intrinsic relationship between
CtCYP82G24-dependant flavonoid accumulation and the movement of
stomatal aperture. Nonetheless, we cannot eliminate other regulatory
and functional genes that could promote PEG-induced metabolite
accumulation and stomatal opening in plants. Thus, more efforts are
still required to clarify the effect of PEG on other functional genes
involved in the flavonoid biosynthetic pathway.
5. Conclusions
In summary, our findings presented the discovery and characterization
of a new CtCYP82G24 isolated from Carthamus tinctorius using an
expressed sequence homology-based approach followed by GFP tagging and
expression analysis in a transgenic system. The result of the
overproduced CtCYP82G24 protein during in vitro enzymatic activity is
strongly associated with flavonoid biosynthesis in transgenic
Arabidopsis. In addition, the differential gene expression level of the
core structural genes of the flavonoid pathway confirmed that the
mediation of CtCYP82G24 might influence the entire flavonoid
biosynthetic pathway of transgenic plants during the early phases of
osmotic stress induction. These findings provide a theoretical and
practical basis for identifying the intrinsic relation of a
stress-responsive cytochrome P450 gene during flavonoid biosynthesis in
plants.
Acknowledgments