Responses of the flavonoid pathway to UV-B radiation stress and the correlation
with the lipid antioxidant characteristics in the desert plant Caryopteris
mongolica
LIU Meiling1,2, CAO Bo1,2, ZHOU Shenghui1, LIU Yubing1
(1.Laboratory of Plant Stress Ecophysiology and Biotechnology, Shapotou Desert Research & Experiment Station, Cold and Arid
Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Donggang West Road 320, Lanzhou, 730000,
China; 2. Graduate University of Chinese Academy of Sciences, Beijing 100049, China)
Abstract: Caryopteris mongolica is a dwarf shrub mainly found in grassland and desert areas of north-west
China, and which can survive severe environmental stress. This study aimed to assess the responses of the
flavonoid pathway to UV-B radiation treatments and its correlation to the lipid peroxide and antioxidant systems in
C. mongolica. In UV-B radiation experiments, plants were exposed to UV-B radiation treatments with a intensity
of 30 J/s for 1, 4 and 24 h, respectively. A control group without UV-B radiation treatment was also used. The
chlorophyll fluorescence parameters, contents of chlorophyll and carotenoid, levels of lipid peroxidation, activities
of antioxidant system enzymes, accumulations of total flavonoids and anthocyanins, and activities of
phenylalanine ammonialyase (PAL) and chalcone isomerase (CHI) under different UV-B radiation treatments
were investigated. The correlations between products and key enzymes in the flavonoid pathway and the lipid
peroxide and antioxidant systems were also analyzed. The results showed that chlorophyll fluorescence parameters
decreased within 24 h of treatment. The chlorophyll contents decreased within 4 h and remained stable after 24 h.
Carotenoid content significantly increased. The level of MDA, the activities of superoxide dismutase (SOD),
ascorbate peroxidase (APX) and peroxidase (POD) and the contents of total flavonoids and anthocyanidins
increased, while catalase activity decreased under UV-B stress. The activities of PAL and CHI also increased with
the increased content of total flavonoids. The flavonoid products anthocuanidins had a significant positive
correlation with MDA level, as well as the activities of antioxidant enzyme SOD. In conclusion, UV-B radiation
induced the degradation of photosynthetic pigments and decreased photochemical efficiency of Photosystem II;
increased the contents of MDA , total flavonoids and anthocyanidins; and also enhanced activities of antioxidant
enzymes (SOD, APX and POD) and key enzymes in the flavonoid pathway (PAL and CHI) in C. mongolica. Thus,
we speculate that the secondary metabolites of the flavonoid pathway were involved in the regulation of stress
*Corresponding author.
E-mail address: [email protected]
resistance in C. mongolica.
Key words: antioxidant enzyme system, flavonoids, Caryopteris mongolica, UV-B radiation stress
1. Introduction
Due to the depletion of ozone layer, elevated UV-B radiation is becoming one of the most
important components of terrestrial radiation that plants are exposed to at the Earth’s surface.
Increased UV-B radiation significantly affects growth, development, morphology, self-protection
and other physiological and biochemical characteristics of plants [1]. It is important to consider the
potential effects of the increased UV-B radiation on photosynthetic systems, activity of enzymes,
membrane systems and secondary metabolism in plants. Studies have indicated that UV-B
radiation can lead to lipid peroxidation and accumulation of MDA[2]. Several studies have
demonstrated that UV-B radiation decrease the growth, chlorophyll content and expression of
genes involves in photosynthesis
[3]。Plants
adapt by using different strategies which play a
protective role against potential damage by UV-B irradiation during their life cycle. UV-B
radiation increase the content of antioxidants and activities of antioxidant enzymes [4,5]。The leaf
thickness and the concentration of phenolic compounds in leaf were found to increase under
enhanced UV-B radiation[6,7]. Enhanced UV-B radiation can induce the expression of flavonoid
pathway genes and accumulation of UV-absorbing flavonoids[8]. In addition to acting as pigments
in plants, flavonoids play a vast array of other biological functions, especially in stress resistence
[9]。
Caryopteris mongolica, a native of Mongolia and western China, is a type of sub-shrub
belonging to Verbenaceae, Caryopteris. C. mongolica can survive in severe extreme environment,
show tolerance to drought, salinity, extreme temperature and UV radiation[10]. Consequently, there
is an interest in studying stress resistance in shrubs. C. mongolica is one of the most promising
plants for such studies. In present paper, we analyzed the correlations between products and key
enzymes in the flavonoid pathway and the lipid peroxide and antioxidant systems under UV-B
radiation of 24 h. According to these studies, we hope to explain the protective role of flavonoids
after UV-B radiation in C. mongolica, to explore the regulation mechanism of flavonoids involve
in the stress resistance further.
2. Materials and methods
2. 1 Materials and treatments
Seeds of C. mongolica were obtained from the Baita foothills of Lanzhou City, Gansu, China.
The seeds were treated with 1% hypochlorite potassium for 10 min, washed with tap water,
soaked in water for 12 h, and then planted in individual 9-L plastic pots containing soil. The pots
were placed in a greenhouse (16/8 h photoperiod; 25℃/12℃ day/night; PAR 150 mol/m2/s,
relative humidity 30%). In the UV-B radiation experiments, plants were exposed to UV-B
radiation treatments with a intensity of 30 J/s for 1, 4 and 24 h, respectively. A control group
without UV-B radiation treatment was also used. The UV-B lamps were made in the Wuxi Jinhua
test equipment company, China, and the irradiation dose was measured with a UV-B radiation
detector produced by Beijing Normal University. Leaves from plants for each treatment were
immediately placed in liquid nitrogen and stored at −80 °C prior to extraction.
.
2.2 Methods
The level of lipid peroxidation in leaf samples was determined in terms of MDA content
according to the method of Zhang[11]. The activities of antioxidant system enzymes were measured
by the methods of Liu[12]. The relative contents of total flavonoids and anthocyanidins were
measured according to the methods of Liu[13]. CHI activity was measured with the method of Li
[14].
PAL activity was measured and calculated by a modified method of Zhang
[11].
The
chlorophyll content and chlorophyll fluorescence parameters were measured according to the
method of Liu[15]. A PAM-2000 chlorophyll fluorometer was used. Analysis of F0 and Fm were
conducted after 30min of dark-adaption.
All data were presented as means ± standard deviations of three determinations. Statistical
analyses were performed using the Student’s t-test and one-way analysis of variance. Multiple
comparisons of means were done by the LSD (least significant difference) test. Statistical
assessments of differences with the same letter between mean values were performed by Duncan’s
multiple range test at P ≤0.05.
3. Results
3.1 Effect on chlorophyll fluorescence parameters and photosynthesis pigments
Chlorophyll fluorescence parameters and photosynthetic pigment content is shown in Figure
1. Under UV-B radiation treatments, little changes in the value of F0, and variations between
different treatments were not significant( figure 1A). The values of Fm decreased as the UV-B
treatment time increased (figure 1B). The values of Fv/Fm (photochemical efficiency of
Photosystem II) displayed decreases, and the variation were significant after 24 h (figure 1C).
A
400
Fo (Relative Unit) )
a
300
a
a
a
200
100
0
B
2500
Fm (Relative Unit))
2000
c
bc
b
1500
1000
500
0
a
C
1
c
b
b
a
Fv /Fm (Relative Unit))
0.8
0.6
0.4
0.2
0
Control
1h
4h
Treatment time
24h
Fig 1. Effects of UV-B radiation treatment (1 h, 4 h, 24 h) on chlorophyll fluorescence parameters in Caryopteris mongolica
Bunge.
Note: (A) Fo; (B) Fm; (C) Fv/Fm. Little letters indicate significant differences (P < 0.05) among the different treatment time under
UV-B expose.
The content of chlorophyll reflects the photosynthesis capacity of plants. The content of
chlorophyll decreased under UV-B stress, and had a negative correlation with treatment time
(figure 2A). Chla, Chlb contents, and the ratio of Chla /Chlb displayed significant decreases
within 4 h (Figure 2B). The content of chlorophyll showed a decline trend within 4 h, and kept
stable after 4 h. The caretonoid content exhibited an increase trend during the experiment (Figure
2A). In the leaves of C. mongholica, the ratio of caretonoids /Chl revealed a significant increase
trend (Figure 2B).
A
Pigment contents (mg/g FW) )
1.4
a
Chla
a
1.2
Chlb
Caretonoids
c
1
0.8
0.6
A
A
b
b
a
B
b
0.4
b
B
0.2
0
Control
1h
Treatment time
4h
24h
B
2.5
a
Chla/chlb
Pigment ratio (%)|
Caretonoid/Chl
a
2
1.5
D
b
C
b
1
B
0.5
A
0
Control
1h
4h
24h
Treatment time
Fig 2. Viriation of pigment contents in Caryopteris mongolica Bunge. under UV-B radiation treatment.
Note: (A)Contents of chlorophyll a, chlorophyll b and carotenoid; (B) Ratio of chlorophyll a/ chlorophyll b and chlorophyll/
caretonoid. The same type letters indicate significant differences (p < 0.05) among the different treatment time under UV-B
expose.
3.2 Effect on MDA and antioxidant enzymes
MDA content is usually used to measure the extent of lipid peroxidation. The content of
MDA were not changed within 1 h of UV-B treatment in C. mongholica. However, the
content of MDA displayed a significant increasing trend after 4 h, and reached to 2 times
higher than control after 24 h (figure 3A). POD(figure 3B), APX (figure 3C), and SOD
(figure 3D) activity increased as the treatment time increade, and showed a significant
increase after 4h. But CAT activity revealed different trend, with a decrease under UV-B
radiation (figure 3E).
A
B 400
c
POD activity (U/min·mg pr) )
MDA content (umol/g FW))
16
14
12
10
b
8
a
6
a
4
c
300
200
a
0
D 10
C 250
c
c
SOD activity (U/min·g pr) )
APX activity (U/min·g pr) )
b
100
2
0
d
200
150
100
b
a
50
b
b
4h
24h
8
6
4
a
a
2
0
0
Control
1h
Treatment time
E
CAT activity (U/min·g pr) )
50
c
40
30
b
b
20
a
10
0
Control
1h
4h
24h
Treatment time
（h）
Fig 3. Effects of UV-B radiation treatments (1 h, 4 h, 24 h) on lipid peroxide and activities of antioxidant enzymes of C. mongolica
Bunge.
Note: (A) malondialdehyde (MDA) content; (B) peroxidase (POD) activity; (C) ascorbate peroxidase (APX) activity; (D)
superoxide dismutase (SOD) activity; (E) catalase (CAT) activity. Little letters indicate significant differences (p < 0.05) among
the different treatment time under UV-B expose.
3.3 Effect on flavonoid
In C. mongholica, the flavonoids metabolism shows positive correlation with the UV-B
treatment time (figure 4). Total flavonoids content(figure 4A), Anthocuanidins(figure 4B)
content and PAL(figure 4C) and CHI (figure 4D) activity were increased as the treatment
time increased. And the values of the samples at 24 h reached to two times of that in control.
B 30
d
c
ANS relative content /g FW)
Total flavonoids content (mg/g FW)))
A 16
12
b
8
b
a
4
20
b
15
c
a
10
5
0
0
c
d
D 25
CHI activity(U/min·mg pr) )
C 20
PAL activity(U/h·mg pr) )
25
16
b
12
a
8
4
c
20
b
15
10
a
b
5
0
0
Control
1h
4h
Treatment ime
24h
Control
1h
4h
Treatment time
24h
Fig 4. Effects of UV-B radiation treatment (1h, 4 h, 24 h) on (A) Content of total flavonoids；(B) Content of anthocyanin；(C)
Phenylalanine Ammonia-lyase (PAL) activity and (D) Chaleone isomerase (CHI) activity in C. mongolica Bunge.
Note：Little letters indicate significant differences (p < 0.05) among the different treatment time under UV-B expose.
3.4 The correlation of flavonoid pathway to the lipid peroxide and antioxidant systems
Under UV-B radiation, in C. mongholica there is a significant correlation between anthocyanidins,
MDA and SOD. The change of anthocyanidins content induced by UV-B radiation may be related
to the extent of lipid peroxidant and SOD activity. The flavonoids content has a positive
correlation to the key enzyme of flavonoids pathway and antioxidant system, but not significant.
Table 1.The correlation coefficient between flavonoid pathway and MDA and antioxidant enzymes in Caryopteris mongolica Bunge.
under UV-B radiation stress.
Note: **correlation is signification at the 0.01 level;*correlation is signification at the 0.05 level.
Correlation coefficient with other physiological and biochemical characters
MDA
POD
APX
SOD
CAT
0.910
0.433
0.505
0.903
-0.486
0.972*
0.844
0.853
1.000**
-0.936
PAL activity
0.803
0.717
0.784
0.803
-0.632
CHI activity
0.864
0.810
0.836
0.870
-0.923
Total flavonoids
content
anthocyanidins
content
4. Discussion
By investigating chlorophyll fluorescence parameters, we found F0、Fm and Fv/Fm
decreased in varying degrees and demonstrated that UV-B stress affects the activity of PSⅡ
and the electron transport. Chlorophyll is the material basis for photosynthesis in plants. The
damage and degradation of chlorophyll could decrease the photosynthetic rate, and
consequently weaken the growth of plants. The chlorophyll content declined under
continuous UV-B radiation, with negative correlation to the treatment time in C. mongholica.
The chlorophyll content markedly reduced in 4 h, but kept stable after 4 h. This demonstrated
that after 4 h of stress, C. mongholica adapt to the dose of UV-B radiation. The caretonoid
content increased with the treatment time. The caretonoid frequently serve as photosynthetic
pigment, but are also endogenous antioxidant, protecting plants from photo-oxidative damage
by scavenging oxygen free radicals in the photo-oxidation process. The ratio of Chla /Chlb
represent the level of photosynthetic activity, while the ratio of caretonoids /Chl involve in
the capacity of tolerance to adversity of plants. During the experiment, the ratio of Chla /Chlb
was decrease. In contrast, the ratio of caretonoids /Chl significantly increased in C.
mongholica. The result indicated the photosynthetic capacity reduced, whereas the resistence
significantly increased under UV-B treatment.
When plant exposes to adversity, MDA accumulate and then crosslink with protein,
nucleic acid, amino acid and other active substances, forming insoluble compounds
(lipofuscin) which impair the structure and function of membrane, increase the leakage of
electrolytes and influence the liquidity of cell membrane and adhesion of enzymes[16].
Consequently, as a product of the per-oxidation of membrane lipid, the MDA content reflects
the level of membrane lipid per-oxidation and extent of injury by environment stress. In this
study, under persistent UV-B radiation, the increasing of MDA indicated the extent of stress
was enhanced.
Antioxidant enzymes play a crucial role in the dynamic balance between the production and
scavenging of reactive oxygen species (ROS). As important compositions of antioxidant enzyme
system in plant cell, SOD, CAT, POD and APX have apparent roles in clearing the ROS in vivo.
This study indicated that SOD, CAT, POD and APX are all involved in the protection against
oxidative stress in C. mongholica. In the present study, SOD, POD, and APX activities in C.
mongholica showed significant increases during the experiment, but CAT activity was slightly
decreased. This may be interpreted as the damage of enzyme protein and inhibition of gene
expression involved in the enzyme protein caused by the UV-B stress. Accordingly, the total
activity of anti-oxidative enzyme system were increased.
Flavonoids are ubiquitous plant secondary metabolites that have a vast array of biological
functions in plants. They play a crucial role in photosynthesis, respiration, growth, reproduction
and other functions in plant stress defense [7,17-18]。The protective property flavonoids display
against UV-B radiation has been shown in a number of studies. Flavonoid composition accumulate
and their antioxidative capacity increase in several plants and callus as a consequence of UV-B
radiation
[19,20]
。In the present work there were increases in the content of total flavonoids,
anthocyanidins and the activities of PAL and CHI in C. mongholica after treatment. The reaction
catalyzed by the key enzyme PAL is the first committed step for the biosynthesis of the
phenylpropanoid skeleton [21]. Perhaps the most stereo-chemically important reaction of flavonoid
biosynthesis is that catalyzed by CHI. (2S)-flavanones which is specifically generated by CHI is a
critical intermediate for formation of several flavonoid classes. By assessing acclimation
responses to four UV regimes by near isogenic maize lines varying in flavonoid content, Casati
and Walbot found that flavonoids can serve as shields against harmful radiation, and the UV-B
radiation can activate additional pathways not shared with other stresses[22]. An investigation about
the impact of UV-B radiation on the soybean leaf proteome suggested that high levels of
flavonoids lead to a reduction in UV-B sensitivity at the proteomic level[23].
There were different degrees of correlation between the flavonoids metabolism and
anti-oxidative enzymes system. anthocyanidins has a significant correlation to the POD activity,
especially to the SOD activity, which indicated that there is a synergic interaction between the
flavonoids and anti-oxidative enzymes which contribute to hostile environment protection
collaboratively. The key enzymes of flavonoids pathway PAL and CHI activity have correlation
with anthocyanidins and total flavonoids and MDA. From the standpoint that membranes is the
target of UV damage, the level of lipid peroxidation represents the extent of stress [24]. The result
in this study demonstrated that the membrane was damaged, and the flavonoids pathway was
induced and play the anti-oxidative property in response to UV-B irradiation. Chelation of both
iron and copper by the carbonyl and
hydroxyl groups of flavonoids prevents lipid peroxidation [25,26]。So flavonoids have been found to
own potent antioxidant activity [27]. It was long supposed that the UV absorptive properties is the
evidence of the protective role of flavonoids [28]. However, in this research, the anthocyanidins
content increased in response to UV-B, and has significant correlation to SOD activity. Several
studies have demonstrated anthocyanidins are UV-A absorbing substances
[29,30].
Thus this
research indicated that anthocyanidins play a anti-oxidative property, not UV absorption,
supporting the suggestion that flavonoids have anti-oxidative role beyond the absorption of UV
radiation [27]. A study in vitro showed flavonoids could inhibit the increase of MDA in liver, kidney
of mice and brain of rabbit, in a dose-dependant relationship, and enhance the activity of SOD [31].
Total flavonoids in Gingo biloba leaf can increase the SOD activity as well
[32]
. The UV-B
radiation experiments conducted by Noriaki showed the antioxidant capacity peaked within 24 h,
and the flavonoids content increased. The anti- oxidative enzymes activity peaked at 6 d
[33].
It
may be explained that under UV-B radiation, a wide variety of free radicals are produced, leading
lipid peroxidation and cell autolysis. At this point, flavonoids contents corresponding increase, or
convert into protective flavonoids, eliminating free radicals, reducing UV-B damage and
enhancing anti-oxidative ability. Whereas in C. mongholica, under enhanced UV-B radiation, the
activity of antioxidant system increased after 4 h, which suggested that flavonoids content does
not represent the endurance ability of plants to UV-B stress. There are two strategies for UV-B
tolerance of flavonoids in plants: under low-dose UV-B stress, the flavonoids accumulate in the
epidermal layers to serve as shields against radiation; under excess UV-B radiation, as the UV-B
which penetrate the epidermal layer increased, special flavonoids are produced to scavenge ROS,
reducing the UV-B damage to plants [34].
Acknowledgments:
This work was financially supported by the National Natural Science Foundation of China
(30800122, 31070358 and 30960065) and the West Light Foundation of the Chinese Academy of
Sciences.
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