Introduction
Many secondary metabolites synthesized
by plants provide some valuable medicinal compounds. Catharanthus roseus (L.)
belongs to the Apocynaceae family and produces more
than 130 Terpene Indole Alkaloids (TIAs).
These include vinblastine (VBL), vincristine (VCR),
vindoline (VIN) and catharanthine (CAT), which have medicinal value. VBL and
VCR can produce effective antitumor compounds used in the
treatment of several types of cancer (Verma et al. 2007).
These alkaloids are in
low contents in leaves and other tissues (Mujib et al. 2014).
Therefore, the alkaloids depend on a large amount
of plant material, tissue cultures and chemical synthesis which are not
economical or feasible to meet the commercial demand (El-Sayed
and Verpoorte
2007). For this reason, field scale cultivation in C. roseus continues
to be the only economic way to alkaloids source (Andrade et al.
2013).
Terpene Indole Alkaloids (TIAs) biosynthesis
pathway has been widely studied and regulated by key
enzymes (El-Sayed and Verpoorte 2007). A number of genes of TIAs metabolic pathway and transcriptional regulators have
been cloned and characterized in C.
roseus (Liu et al. 2017). The high expression
of genes encoding
some TIA pathway key enzymes cannot
be completely increased alkaloids production (Pollier
et al. 2014). A very promising method is to
enhance the expression of several transcription factors (TFs) regulating the TIA pathway and enhance alkaloid production
(Andrade et al. 2013). It has been reported that
the expression of Myc1 has positive
regulation effect in secondary metabolic synthesis during environmental stress
(Guo et al. 2016). Myc1 is a basic transcription factor
containing a conserved domain of the helix-loop-helix which belongs to the
transcription enhancer.
Secondary metabolites are
synthesized or induced by various developmental, hormonal and environmental
factors (Liu et al. 2017). Evidence has been obtained that the TIAs
accumulation in the plants of C. roseus was related to environmental factors (Xiao et al.
2013; Zhu et al. 2016). As a signal of plant morphogenesis, light
strongly affects the secondary metabolism of plants.
It has been reported that plant growth and the production of these metabolites
compounds under light-shading environment can be
significantly promoted (Lamattina et al.
2003). Panda et al. (2011) have reported that exogenous nitric
oxide (NO) can alleviate various stresses in plants. NO is a free
radical and takes part in many plant physiological processes (Yu et al.
2005) including plant growth, development, and defense responses (Xiong et al. 2010). Recently, the relation of NO and plant growth hormones has been reported (Shen
et al. 2013), but little information is available
about the
changes of plant hormones and
TIAs biosynthesis accumulation under the light shading stress. The
present study evaluated the relation of exogenous NO and the secondary
metabolism in C. roseus under light-shading environment.
Materials
and Methods
Plant
materials and treatments
The study plant for experiment was C. roseus, and plants were exposed
light-shading treatments at 45 days after sowing while the sun shading rate was
55–60%. A uniform concentration (0.01 mmol·L-1)
of sodium nitroprusside (SNP) was sprayed to soil surface; water spray of equal
volume was used as a control. Treatments included the following: normal growth
as control; application of SNP; light-shading; light-shading and with
application of SNP, and the similar plants were treated by different conditions
for 7 days. Three replicates were analyzed. Fresh leaves samples were frozen in
liquid nitrogen and immediately kept at -80°C for further analyses.
Determination of plant growth
and yield
The
plants for growth assay were uprooted and carefully washed and adhered water
particles were removed from roots. Then the fresh weight per plant, and length
of shoot and leaf was measured. The plants were dried for 24 h with at 75°C)
for dry weight. Leaf-area index (LAI) was measured by using the following
formula as suggested by Watson (1947).
LAI =Leaf-area per plant/soil area occupied by plant
Determination of plant hormones content
The plant hormones including IAA, 6-BA and ABA contents
were determined by high performance liquid chromatography (HPLC) method. The hormones
from fresh leaves were extracted by methanol. The chromatographic conditions
used were as follows: C18 silica gel column (250 mm × 4.6
mm, 7 μm),
mobile phase: methanol-0.6% acetic acid (V:V=50:50),
column temperature: 35°C, flow rate: 1 mL / min, volume flow rate: 10 μL,
wavelength: 254 nm.
Estimation of alkaloids content
The analysis for alkaloids contents (Vindoline, VIN;
Catharanthine, CAT; Vinblastine, VBL and Vincristine, VCR) in leaves, were
performed by HPLC (Jasco, VG, England). The methanol extracts were dried at 50°C
in a rotary evaporator. The chromatographic conditions were as follows: C18
silica gel column (250 mm × 4.6 mm, 7 μm), the mobile phase A was the mixture of deionized
water and diethylamine (VH2O: Vdiethylamine= 990:10, pH 7.3 with phosphoric
acid); the mobile phase B was methanol, column temperature: 28°C, flow rate:
1.2 mL/min, volume flow rate 10 μL, wavelength: 220 nm.
RNA isolation and quantitative real-time PCR (RT-qPCR)
Total RNA was
extracted from the leaf samples using TRIzol reagent
(Sangon, Shang hai, China),
and was stored at -80oC. The first-strand cDNA synthesis was
performed by using the BeyoRT™ II cDNA
(Beyotime, Nanjing, China), and was used in RT-qPCR
reactions. Each reaction contained a mixture of 1 μL cDNA aliquots, 1.5 μL of mixed primers, 5 μL of BeyoFast™ SYBR Green qPCR Mix (Beyotime, Nanjing,
China) and 11 μl nuclease-free water.
The list of primer pairs used for the RT-qPCR is shown in Table 1. The RT-qPCR amplification was carried out in 96-well
plates on a LightCycler® 480II System (Roche, Switzerland Roche Diagnostics, Indianapolis, I.N.,
U.S.A.). The cycling
parameters consisted of a 95ºC hold for 3 min, followed by 40 cycles of a 95°C denaturing
step for 15 s and a 49–52.5oC annealing/extension step for 30 s, and
PCR reactions with water were also carried out as negative controls. Each primer
analyzed was performed in triplicate, the relative gene expression was
quantified according to Schmittgen and Livak (2008) method.
The primer of Ribosomal 40S protein S9 (RpS9) was used (Li et
al. 2015) as internal control.
Statistical analysis
The data obtained were analyzed using Excel 2007 and S.P.S.S.
19, and the differences in the expression levels were tested by Duncan’s
multiple-range test. All the results were replicated three times, respectively.
Results
Growth
and yield
There was significant increasing in growth traits in
contrast to the CK, the change trend was light-shading and with application of
SNP > light-shading > application of SNP > control; the change trend
of node spacing, stem diameter and fresh weight was
light-shading and with application of SNP > application of SNP > light-shading
> control (Table 2). Compared with CK, the treatment of light-shading and
with application of SNP significantly promoted the growth index of C. roseus, which indicated that
exogenous NO significantly regulated the growth and development of C. roseus
under shading stress.
Plant hormones content analyses
The IAA, 6-BA and ABA contents were increased
significantly (P < 0.01) in comparison to CK that
showed the exogenous NO increased hormone contents in the treatment of
application of SNP and light-shading and with application of SNP. Under shading
stress, the ABA content only increased significantly (P < 0.05) in comparison to CK. Plant hormone
might improve plant metabolism, led to enhanced plant growth and production
(Fig. 1).
Table 1: The
primers were used in this work
|
Primer |
Primer sequences
(5’-3’) |
Amplified size (bp) |
Gene ID |
|
Myc1 |
CCT CAT TCA TGG CAT TGG C |
250 |
AF283506.2 |
|
GTT TCC GAT GAA CAG CGC TAC |
|||
|
40S |
GGT TGT CAA TGT TCC TTC CTTC |
167 |
AJ749993.1 |
|
TCT TCA TCC TCT TCA TCT CCA
TC |
|||
|
G10h |
GTA CAG GAA CTA ATT GCG TAT
TGC |
106 |
AJ251269 |
|
CGA CGT CAA CCG CTT CTC |
|||
|
Tdc |
AAA ATG TTC GAA GAA TGG GTT AGA |
109 |
X67662 |
|
GTT TCT CGG TAC CAC AAT TTC
G |
|||
|
Str |
TGT GAG AAC AGC ACC GAT CC |
157 |
X53602 |
|
TTG TGG CTA GTTGTG TGG CA |
|||
|
Sgd |
CAT TGG TGA ACC GTG CTA TG |
121 |
EU072423 |
|
AGA TTG TAG AGT CCA GAT GGA
ACA |
|||
|
Dat |
CAC
GGT ATC AGG GAA ATC AG |
142 |
AF053307 |
|
CTG GAA ATG GCA AAG ATT GG |
Table
2: Effects on growth morphological indexes of C. roseus seedlings
|
Treatments |
Control |
Application of SNP |
Light-shading |
Light-shading + SNP |
|
Plant height (cm) |
15.40 ± 1.33a |
16.80 ± 1.93a |
17.40 ± 1.29a |
20 ± 1.58b |
|
6.67 ± 0.67a |
7.67 ± 1.45a |
7.33 ± 0.67a |
8.67 ± 1.76b |
|
|
Leaf length (cm) |
6.56 ± 0.16ab |
5.78 ± 0.25b |
6.06 ± 0.43ab |
6.90 ± 0.41a |
|
Leaves wide (cm) |
2.40 ± 0.19a |
2.32 ± 0.11a |
2.34 ± 0.14a |
2.50 ± 0.08a |
|
2.79 ± 0.18a |
2.51 ± 0.16a |
2.60 ± 0.14a |
2.77 ± 0.19a |
|
|
Leaf area (cm2) |
9.20 ± 1.11a |
9.00 ± 0.45a |
8.80 ± 0.49a |
9.40 ± 0.6a |
|
Node spacing (cm) |
0.92 ± 0.04c |
1.18 ± 0.07ab |
1.08 ± 0.04b |
1.30 ± 0.06a |
|
Stem diameter (cm) |
0.25 ± 0.02c |
0.34 ± 0.02b |
0.30 ± 0.01bc |
0.52 ± 0.02a |
|
Whole plant fresh weight (g) |
3.76 ± 0.07b |
3.38 ± 0.04bc |
2.96 ± 0.05d |
5.36 ± 0.26a |
|
Whole plant dry weight (g) |
0.62 ± 0.03b |
0.60 ± 0.03b |
0.55 ± 0.04b |
0.80 ± 0.1a |
Different lower case letters
indicate significant differences (P < 0.05) T1, application of SNP;
T2, light-shading; T3, light-shading and with application of SNP. Same as below
Alkaloids content
Vindoline (VIN), Catharanthine (CAT),
Vinblastine (VBL) and vincristine (VCR) contents in the leaves
were slightly increased under application of SNP and
light-shading treatment, but the VCR content significantly changed. The
contents of VIN and CAT increased significantly (P < 0.01) under light-shading and with application of SNP
treatment, which were 4.65 and
6.13 times compared to control, respectively. The contents of VBL and VCR
also increased significantly (P < 0.05),
which were 2.48 and 1.64 times compared to control, respectively (Fig. 2a–d).
TIA biosynthetic genes mRNA levels
The expression of transcription factor (Myc1) was down-regulated (P
< 0.05) significantly under application of SNP and light-shading treatment (Fig. 3a) in comparison to control, and up-regulated (P < 0.05) significantly under
light-shading and with application of SNP treatment (Fig.
3a). It showed that the expression of the transcription factor was
activated under compound stress (light-shading and with application of SNP). In
the treatment of light-shading and with application of SNP, the expression of
G10h, Tdc and Str
were significantly up-regulated (P <
0.05) (Fig. 3b–f). It indicated that compound treatment can promote the high
expression of the upstream pathway genes during
alkaloid synthesis. In the treatment of application of SNP, the expression of Sgd and Dat increased
significantly (P < 0.05),
suggesting that exogenous NO promoted the high expression of downstream pathway
genes. In a word, the exogenous NO significantly regulated the expression of TF
and key genes in the pathway under light-shading stress.
Correlation analysis
Correlation analysis between hormones content, alkaloids
content and key genes expression in leaves of C. roseus seedlings showed that there was no correlation between Myc1 expression and plant hormone accumulation in
the treatment of control (Table 3); There was a negative correlation
between plant hormone accumulation and Myc1 expression,
between Myc1 expression and Tdc, Str,
Sgd and Dat in the
treatment of application of SNP and light-shading (Table 3). But there was a significant positive correlation between 6-BA
and ABA content and Myc1 expression (P < 0.01) in the treatment of
light-shading and with application of SNP (Table 3), and there was a
significant positive correlation between Myc1 expression and the expression of Tdc, Str, Sgd and Dat (P < 0.01) (Fig. 4). It has been revealed that Myc1 were almost
negative regulators in the treatment of application of SNP and light-shading,
whereas Myc1 were positively regulators (marked in coarse arrow) in the
treatment of light-shading and with application of SNP (Fig. 4). Furthermore,
there was a significant positive correlation (P < 0.01) between the expression of Tdc,
Str and the CAT content (Table 3) in the treatment of
light-shading and with application of SNP, between Dat expression and the VIN content, and between Sgd expression and the content of VBL and VCR. These
results suggested that hormones, as endogenous signals, induced the expression
of Myc1, regulated the high expression of key genes in the metabolic pathway of
alkaloids, and promoted the synthesis of VBL and VCR (Fig. 2).
Table 3: Correlation
coefficient (r) between accumulation of alkaloids, plant hormones and genes
expression
|
Hormone |
Control |
Application of SNP |
Light-shading |
Light-shading + SNP |
||||||||||||||||||||
|
Myc1 |
G10h |
Tdc |
Str |
Sgd |
Dat |
Myc1 |
G10h |
Tdc |
Str |
Sgd |
Dat |
Myc1 |
G10h |
Tdc |
Str |
Sgd |
Dat |
Myc1 |
G10h |
Tdc |
Str |
Sgd |
Dat |
|
|
6-BA |
|
|
|
|
|
|
-0.985** |
|
|
|
|
|
|
|
|
|
0.813* |
|
0.983** |
|
|
|
|
|
|
IAA |
|
|
|
|
|
|
|
|
|
|
|
|
-0.996** |
|
|
|
|
|
|
|
|
|
|
|
|
ABA |
|
|
|
|
|
|
-0.988** |
|
|
|
|
|
-0.998** |
|
|
|
|
|
0.977** |
|
|
|
|
|
|
CAT |
|
|
|
|
|
|
|
|
|
0.854* |
0.876* |
0.861* |
|
|
|
0.814* |
0.901* |
0.842* |
0.992** |
0.830* |
0.986** |
0.997** |
0.999** |
0.999** |
|
VIN |
|
|
|
|
|
|
|
-0.865* |
|
|
0.864* |
0.875* |
|
-0.861* |
-0.820* |
0.850* |
0.858* |
0.883* |
0.987** |
0.845* |
0.984** |
0.994** |
0.997** |
0.999** |
|
VBL |
|
|
1.000** |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
0.975** |
0.862* |
0.991** |
0.984** |
0.993** |
0.992** |
|
VCR |
|
|
|
|
|
|
|
-0.858* |
|
0.874* |
0.872* |
0.875* |
|
|
|
|
|
|
0.982** |
|
0.955** |
0.976** |
0.975** |
0.966** |
**Significant correlation at P < 0.01, * Significant correlation at P < 0.05
%201520-1526_files/image002.jpg)
Fig.
1: Effects on the contents of
6-BA, IAA and ABA under four different treatments in C. roseus
leaves (T1, application of SNP; T2, light-shading; T3, light-shading and with
application of SNP. Same as below)
Data points are mean ± SD of three biologically independent experiments. ** Significant differences with P < 0.01,
* Significant differences with P < 0.05,
same as below
%201520-1526_files/image004.jpg)
Fig.
2: Effects on alkaloids
content in C. roseus leaves under four
treatments
%201520-1526_files/image006.jpg)
Fig. 3: The expression changes of Myc1 and the TIA pathway genes were
detected under the four treatments (40s as internal control, CK as control)
%201520-1526_files/image008.jpg)
Fig. 4: Regulation
of TIA biosynthetic pathway genes by transcription factor in C. roseus
Discussion
As an internal factor, plant
hormones play an important role in regulating plants growth
and development. plant hormones regulated the genes
expression through the modification of transcription and/or translation, and
determined plant growth orientation, physiology, and productivity (Alam et al. 2012). Previous
studies have shown that 6-BA, as a cytokinin, promoted
the differentiation and growth of multiple tissues, and had a synergistic
effect with IAA (Chen et al. 2004). Similarly, Zhang et al. (2018)
reported that ABA content could regulate the synthesis
of indole alkaloids by promoting the expression of CrTdc, CrNmt and CrD4h in C. roseus. The plant hormones content changes manipulated secondary
metabolism and affected alkaloids accumulation in the specific tissue
(Srivastava and Srivastava 2007). Recently, many articles have reported that
there is the relativity of exogenous NO treatment and
the plant hormones content changes (Shen et al. 2013). In this study,
the changes of the content of ABA, 6-BA and IAA were
analyzed under the condition of light-shading + SNP which increased the ABA,
6-BA and IAA contents, as it was beneficial to increase the resistance of
plants and regulate the growth, development and biomass (Table 2), and promote
the synthesis of alkaloids.
The alkaloids biosynthesis is directly regulated
at the transcriptional level. Evidence has been shown that transcription factors
(TFs) play a critical role in regulating the synthesis of TIAs in C. roseus
(Zeng et al. 2017). Myc1 expression could be up
regulated by the induction of jasmonate (JA) or
fungal elicitor and activate the transcription of Str
and Tdc (Verma et al.
2012). There is little doubt that TIA biosynthesis does benefit from the key
genes expression in biosynthesis pathway (Liu et al. 2011). Further
studies have shown that that the Tdc expression was positively correlated with the content of
CAT; Str expression was involved in VIN biosynthesis (Pandey et al.
2016). The mechanisms for promoting alkaloid
synthesis include the combination of transcription factors and specific
elements and/or regulation of the corresponding genes expression. This
study reported that there was a transcriptional regulatory network of TFs in
TIA biosynthetic pathways, and a significant enhancement in the alkaloid
production under the compound treatment
(light-shading and with application of SNP). It is a positive role for plant hormones (6-BA and ABA) in regulation of Myc1
expression, because TFs was usually regulated by signaling molecules or other
elements (for example, plant hormones) (Gao et al. 2015), and then could
activate the expression of Tdc and Str, there was a significantly positive correlation (P < 0.01) between the genes (Tdc and Str) expression and the
alkaloids content (Table 3), then VBL and VCR content accumulation could be
promoted.
Conclusion
This
study evaluated that application of light-shading and SNP improved considerably
alkaloids contents. The role of Myc1
transcription factor (TF) in plant hormones signaling and plant alkaloid
biosynthesis had been systematically analyzed in C. roseus. Notably,
there was a close correlation between NO and plant hormones by inducing high expression of Myc1 gene, then high expression of TIA
synthetic genes, such as Tdc˴Str˴Sgd and Dat, promoted TIA biosynthesis.
Acknowledgements
The work was supported by the Foundamental Research
Funds for the Central Universities (No. 2572019BU01, 2572017BA07); the National Key Research and Development
Program of China (No. 2017YFD0600706) and Heilongjiang Province Natural Science
Fund Project (No. QC2017009)
Author Contributions
Yu-jie Fu and Shu-ping Guo; methodology, Ying Liu;
software, Chang Yang; validation, Yu-jie Fu, Hai-long Weng and Shu-ping Guo;
formal analysis, De-wen Li; investigation, Ying Liu and Chao Yang; data
curation, De-wen Li; writing—original draft preparation, Ying Liu;
writing—review and editing, De-wen Li; supervision, Yu-jie Fu; project
administration, Ying Liu; funding acquisition, De-wen Li and Ying Liu. All
authors have seen the manuscript and approved to submit to your journal.
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